Engineered tissue constructs

ABSTRACT

A modular engineered tissue construct includes a plurality of fused self-assembled, scaffold-free, high-density cell aggregates. At least one cell aggregate includes a plurality of cells and a plurality of biocompatible and biodegradable nanoparticles and/or microparticles that are incorporated within the cell aggregates. The nanoparticles and/or microparticles acting as a bulking agent within the cell aggregate to increase the cell aggregate size and/or thickness and improve the mechanical properties of the cell aggregate as well as to deliver bioactive agents.

RELATED APPLICATION

This application is a Continuation-in-Part of PCT/US2015/019541, filedMar. 9, 2015, which claims priority from U.S. Provisional ApplicationNo. 61/949,552, filed Mar. 7, 2014. This application also claimspriority to U.S. Provisional Application Serial No. 62/384,400, filedSep. 7, 2016, the subject matter of which are incorporated herein byreference in their entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant Nos.R01AR063194, T32AR007505, awarded by The National Institutes of Health,and DGE1144804 awarded by the National Science Foundation. The UnitedStates government has certain rights to the invention.

TECHNICAL FIELD

The present application relates to engineered tissue constructs, andsystems and methods of forming the engineered tissue constructs.

BACKGROUND

Narrowing or collapse of the trachea is a life threatening conditionbecause stenosis or malacia can prohibit sufficient air transport to thelungs. The most common cause of adult tracheal stenosis is trauma due toprolonged intubation or tracheostomy, but other causes includeperichondritis, chondritis, tumor, burns and external trauma. Typically,if the affected portion is less than half the entire length of thetrachea in adults or one third in children, the diseased region can beresected and the healthy ends anastomosed during tracheal reconstructionsurgery. While short, resectable stenoses are far more common, there arelimited treatments available for lengthy tracheal occlusions. Short-termsolutions for patients with long segment stenosis include stents,T-tubes, laser surgery and airway dilation. However, a major drawback tothese is the need for repetitive treatment: periodic stent and tubereplacement due to granuloma formation or additional laser surgery anddilation due to scarring and restenosis. As a result, biomaterial andtissue engineering approaches have been pursued to develop trachealsubstitutes. A functional tracheal replacement must first and foremostmaintain airway patency during normal breathing. Normally, healthytracheal cartilage supports the open windpipe. Acellular trachealprostheses are made of rigid materials and tissue engineered cell-ladentechnologies are typically cartilaginous structures that are designed tomimic the trachea. A variety of tracheal replacement strategies havebeen explored, including cell-free artificial prostheses, autografts,native or decellularized allografts which are often seeded with therecipient’s cells, and autologous de novo tissue engineered constructs.Despite the broad range of approaches, each has shortcomings. Acellulartracheal prostheses often result in tissue granulation, implantmigration, progressive scar tissue formation and restenosis. Autograftsand allografts have limited availability, poor mechanical properties,and undergo remodeling upon implantation, often leading to collapse,scarring, and airway occlusion. Allogeneic donor tissue also carries arisk of disease transmission and immunogenicity; recipients of nativetissues must be immunosuppressed and extra care must be taken to removeantigens from decellularized tissues. Tissue engineered constructscomprised of autologous cells in scaffolds circumvent immune responseissues, but the structural, physical and biochemical properties of thescaffold must be carefully designed to guide cell behavior and neotissueformation. It is also challenging to tune the scaffold degradation rateto match that of cell proliferation and new extracellular matrix (ECM)production, and biomaterial degradation byproducts may hinder tissuehealing.

SUMMARY

Embodiments described herein relate to engineered tissue constructs withdefined shapes, such as engineered tissue rings, systems and methods offorming modular engineered tissue constructs and to the use of modularengineered tissue constructs in modular tissue assembly systems fortissue repair and bio-artificial tissue engineering applications, suchas engineered trachea constructs. The engineered tissue constructs caninclude self-assembled, scaffold-free, cell aggregates that are formedby culturing a plurality of cells and nanoparticles and/ormicroparticles in wells with defined shapes, e.g., rings, disks, orblocks, of a cell culture apparatus or bioreactor. In some embodiments,the self-assembled, cell aggregate can include a population or pluralityof cells and plurality of nanoparticles and/or microparticles that areincorporated within the cell aggregate. The nanoparticles and/ormicroparticles can act as a bulking agent within the cell aggregate toincrease the cell aggregate size and/or thickness. Incorporation of thenanoparticles and/or microparticles in the cell aggregate can alsoimprove the mechanical properties of the cell aggregate formed from thecells and nanoparticles and/or microparticles allowing the cellaggregate to be readily manipulated and formed into engineered tissueconstructs.

In some embodiments, the nanoparticles and/or microparticles can includeat least one bioactive agent that is differentially and/or controllablyreleased by the nanoparticles and/or microparticles. In someembodiments, the bioactive agent can be physically associated with thenanoparticles and/or microparticles and spatially and/or temporallyreleased with a defined release profile from the nanoparticles and/ormicroparticles.

The engineered tissue constructs can be used in a tissue assembly systemto engineer human tissue containing, for example, engineeredcartilaginous, vascular, prevascular, muscular, and bone segments. Thetissue assembly system can permit fusion of engineered tissue constructshaving different properties together to generate modular constructs withmultiple types of tissues in a spatially-controlled pattern. Culture ofheterogeneous engineered tissue constructs in a hollow organ bioreactorcan permit further modification of the constructs, including, forexample, epithelialization, of a surface of the construct.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a method of forming an engineeredtissue construct in accordance with an embodiment.

FIG. 2 is a flow diagram showing a method of forming an engineeredtissue construct.

FIGS. 3(A-D) illustrate images of heterogenous cartilage tube withcartilaginous and non-cartilaginous portions. Chondrogenesis was inducedin hMSC rings for (A-B) 5 days or (C-D) 12 days. SMC rings were culturedfor (A-B) 10 days or (C-D) 5 days. Alternating hMSC and SMC rings werefused into (A-B) 10-ring tubes over 7 days or (C-D) 4-ring tubes over 8days. (A) Fluorescence (hMSC labeled with red cell tracker dye) and (B)brightfield images of hMSC-SMC tubes. (C) hMSC-SMC tube after 8 days offusing. (D) Cross-section of hMSC-SMC tube stained with Safranin O /Fast Green for GAG (lumen is on bottom). Scale bars: white=1 mm,black=500 µm.

FIG. 4 is a schematic of tissue ring and tube assembly processes. Asuspension of MSCs with growth factor loaded microspheres (“hMSC + MS”)was seeded in custom agarose wells and cultured in basal pellet media.Cell only tissues (“hMSC”), which did not contain microspheres, wereseeded and cultured in basal pellet media supplemented with TGF-β1. Onday 2 of culture, rings (tan) were removed from the culture wells usingtweezers and were stacked on silicone tubes (gray) to form 3- and 6-ringtubes.

FIG. 5 illustrates a light photomicrograph of crosslinked gelatinmicrospheres.

FIGS. 6(A-D) illustrate macroscopic images of cartilage tissue rings andtubes in culture. Tissue rings were formed by seeding a suspension of(A) hMSCs or (C) hMSCs with microspheres in custom designed agaroseannular wells (white dotted outline) with 2 mm posts (black dottedoutline). (B, D) On Day 2, some of the rings were stacked on siliconetubes, which were clamped in a custom designed holder, to form 3-ring(white arrow) or 6-ring (black arrow) tissue tubes. MS = Microspheres.

FIGS. 7(A-G) illustrate gross macroscopic pictures of (A-C) hMSC-onlycartilage rings and tubes, (D-F) microsphere-containing rings and tubes,and (G) native rat trachea. (A, B, D, E) Rings and 3-ring tubes areshown in replicates. (C, F, G). A representative hMSC or hMSC + MS6-ring tube and a rat trachea are presented from multiple perspectives.MS = Microspheres. Scale bar is 2 mm.

FIGS. 8(A-C) illustrate (A) DNA content, (B) GAG content, and (C) GAGnormalized to DNA in harvested rings and 3-ring cartilage tubes. MS =Microspheres. Groups that do not share the same letter are significantlydifferent (p < 0.01).

FIGS. 9(A-E) illustrate photomicrographs of Safranin O/Fast Greenstained tissue engineered (A, C) cartilage rings and (B, D) cartilagetubes composed of (A, B) hMSCs-only and (C, D) hMSCs + MS, and (E) rattrachea in axial and vertical planes. Remnant gelatin microspheres(black arrows) are visible in hMSC + MS tissues. MS = Microspheres; F =axial plane; z = vertical plane; black scale bars = 500 mm; white scalebars = 100 mm.

FIGS. 10(A-D) illustrate photomicrographs of Collagen Type II/Fast Greenstained tissue engineered (A, C) cartilage rings and (B, D) cartilagetubes composed of (A, B) hMSCs-only (A, B) and (C, D) hMSCs + MS inaxial and vertical planes. Type II collagen rich tissues are red. MS =Microspheres; F = axial plane; z = vertical plane; black scale bars =500 mm; white scale bars = 100 mm.

FIGS. 11(A-B) illustrate ring thicknesses (A) and tube outer diameters(B) measured in cartilage tissue engineered rings and tubes, and rattracheas. MS = Microspheres. Groups that do not share a letter aresignificantly different (p < 0.05).

FIGS. 12(A-D) illustrate mechanical analysis of tissue engineeredcartilage rings and tubes, and rat tracheas. (A) Ring maximum load atfailure and (B) ultimate tensile stress during uniaxial testing (A,inset); (C) tube load at 80% collapse and (D) % recoil after luminalcollapse (C, inset). MS = Microspheres. Groups that do not share thesame letter are significantly different (p < 0.05).

FIG. 13 is an image showing repeated manual compression and release of arepresentative hMSC tube, hMSC + MS Tube and a section of a rat trachea.MS = Microspheres.

FIGS. 14(A-B) illustrate images showing gross morphology of hMSC ringsand tubes. Representative rings (2-mm) and tubes (3×2-mm and 8×2-mm) ofA) hMSCs alone or B) hMSCs + GM + MCM cultured for 5 weeks inchondrogenic + exogenous TGF-β1 (2 weeks; 10 ng/ml) and osteogenic +BMP-2 (3 weeks; 100 ng/ml) induction media. Scale bar = 2 mm.

FIGS. 15(A-F) illustrate graphs showing the thickness and lengthanalyses of hMSC osteogenic or bone rings and tubes generated using themethods described herein. A, B) 2-mm rings (N=4), C, D) 3×2-mm tubes(N=2), E, F) 8×2-mm tubes (N=1). hMSCs alone or hMSCs + GM + MCMcultured for 5 weeks in chondrogenic + exogenous TGF-β1 (2 weeks; 10ng/ml) and osteogenic + BMP-2 (3 weeks; 100 ng/ml) induction media.

FIGS. 16(A-D) illustrate a schematic view of microsphere incorporationwithin self-assembled vascular cell rings. A, Cross-linked gelatinmicrospheres were mixed in suspension with smooth muscle cells at 0, 0.2or 0.6 mg microspheres per million cells. B, cells and microspheres wereseeded into agarose molds. Cells aggregate to form self-assembled tissuerings with incorporated microspheres (C). D, Photograph of an agarosemold with three aggregated cell-microsphere rings cultured for 14 days.Arrowheads point to self-assembled tissue rings on agarose posts (2 mmpost diameter).

FIGS. 17(A-D) illustrate images and a graph showing microspheresincreased tissue ring thickness. Images of self-assembled vascular ringsseeded with 0 (A), 0.2 (B), or 0.6 mg (C) of microspheres per millioncells and cultured in smooth muscle growth medium for 14 days. (D)Average wall thicknesses of 14-day-old tissue rings with 0, 0.2, or 0.6mg microspheres per million cells. Scale = 1 mm, n = 6, *p<0.05.

FIGS. 18(A-L) illustrate images showing gelatin microsphereincorporation and degradation within vascular tissue rings. Tissue ringswere seeded with 0, 0.2, or 0.6 mg microspheres per million cells,collected at 7 or 14 days, and stained with Hematoxylin and Eosin (A-F)and Picrosirius Red/Fast Green stain. Scale = 100 µm.

FIGS. 19(A-D) illustrate graphs showing mechanical properties ofvascular tissue rings loaded with gelatin microspheres. Self-assembledcell rings were cultured for 14 days in growth medium and pulled tofailure. Mean values for ultimate tensile strength (UTS; A), maximumtangent modulus (MTM; B), failure load (C) and failure strain (D) werecalculated from stress-strain curves for each ring sample group. n = 6,*p<0.05.

FIGS. 20(A-D) illustrate an image and graph showing microspheresincreased ring thickness in tissues cultured in smooth muscledifferentiation medium. Rings were seeded with 0 (A), 0.2 (B), or 0.6 mg(C) of microspheres per million cells and cultured to 14 days. Ringswere seeded in growth medium and switched to differentiation medium onday 1. (D) Average wall thicknesses of 14-day-old tissue rings with 0,0.2, or 0.6 mg microspheres per million cells. Scale = 1 mm; n = 8 for 0mg; n = 9 for 0.2 and 0.6 mg/million cells; *p<0.05.

FIGS. 21(A-L) illustrate images showing microsphere incorporation withintissue rings cultured in smooth muscle differentiation medum. Tissuerings were seeded in growth medium with 0, 0.2, or 0.6 mg microspheresper million cells, and switched to differentiation medium at day 1.Tissue rings were collected at 7 or 14 days, and stained withHematoxylin and Eosin (A-F) and Picrosirius Red/Fast Green stain. Scale= 100 µm.

FIGS. 22(A-D) illustrate graphs showing the mechanical properties oftissue rings cultured in differentiation medium with microsphereincorporation. Self-assembled cell rings were seeded in growth medium,switched to differentiation medium on day 1, and cultured for 13 days indifferentiation medium and harvested for mechanical tests (14 days totalculture). Mean values for ultimate tensile strength (UTS; A), maximumtangent modulus (MTM; B), failure load (C) and failure strain (D) werecalculated from stress-strain curves for each ring sample group. n = 6,*p<0.05.

FIGS. 23(A-G) illustrate images and a graph showing exogenous ormicrosphere-mediated TGF-β1 delivery to self-assembled tissue rings.Rings were seeded in growth medium, and switched to differentiationmedium at day 1. (A) Untreated control rings with no microspheres (n=6).(B) Tissue rings treated with 10 ng/ml soluble TGF-β1 (n=8). (C,D)Tissue rings with unloaded gelatin microspheres (0.6 mg/million cells;n=6) untreated (C) or treated (D) with 10 ng/ml exogenous TGF-β1 (n=8).(E) Tissue rings with microspheres pre-loaded with TGF-β1 (400 ngTGF-(31/mg microspheres), but no exogenous TGF-β1 in the medium (n=7).Tissue rings contracted after they were removed from agarose posts,resulting in a greater decrease in diameter (F) and greater thickness(G) in rings exposed to TGF-β1. Scale = 1 mm, *p<0.05.

FIGS. 24(A-J) illustrate images showing hematoxylin and eosin stain(A-E) at 14 days shows microsphere degradation primarily in the groupswith added TGF-β1. Collagen deposition in TGF-β1 groups is primarilyseen around ring edges. (A, F) Control (untreated) rings. (B, G) Ringscultured with exogenous 10 ng/ml TGF-β1 added to the medium. Rings withunloaded microspheres (0.6 mg per million cells) untreated (C, H) ortreated with 10 ng/ml exogenous TGF-β1 (D, I). Rings with TGF-β1 loadedmicrospheres (0.6 mg microspheres per million cells) and no exogenousTGF-β1 treatment (E, J). Scale = 100 µm.

FIGS. 25(A-J) illustrate images showing contractile protein expressionin tissue rings treated with TGF-β1. Rings were grown with either nomicrospheres or exogenous TGF-β1 (A, F), no microspheres but treatedwith 10 ng/ml exogenous TGF-β1 (B, G), with microspheres and noexogenous TGF-β1 (C, H), with unloaded microspheres and exogenous TGF-β1(D, I) or with TGF-β1 loaded microspheres and no exogenous TGF-β1 in themedium (E, J). Rings were stained for either smooth muscle alpha actin(A-E) or calponin (F-J). Nuclei are shown in blue (Hoechst). Scale = 100µm.

FIGS. 26(A-D) illustrate graphs showing the mechanical properties oftissue rings treated with TGF-β1 after 14 days in culture. Rings werecultured in differentiation medium with no microspheres or exogenousTGF-β1, no microspheres with 10 ng/ml exogenous TGF-β1, unloadedmicrospheres with no exogenous TGF-β1, unloaded microspheres with 10ng/ml exogenous TGF-β1, or loaded microspheres (400 ng TGF-β1/mgmicrospheres) and no exogenous TGF-β1. Rings in the group with nomicrospheres and exogenous TGF-β1 had significantly higher ultimatetensile stresses than the loaded microsphere group (A), and the unloadedmicrospheres without TGF-β1 group had significantly higher failure loadsthan rings without microspheres or TGF-β1 (B). There were no significantdifferences in MTM (C) or failure strain (D). *p<0.05.

DETAILED DESCRIPTION

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises, such as Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates). Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thepresent invention pertains. Commonly understood definitions of molecularbiology terms can be found in, for example, Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th Ed., Springer-Verlag: New York,1991, and Lewin, Genes V, Oxford University Press: New York, 1994. Thedefinitions provided herein are to facilitate understanding of certainterms used frequently herein and are not meant to limit the scope of thepresent invention.

As used herein, the term “autologous” refers to cells or tissues thatare obtained from a donor and then re-implanted into the same donor.

As used herein, the term “allogeneic” refers to cells or tissues thatare obtained from a donor of one species and then used in a recipient ofthe same species.

In the context of the present invention, the term “bioactive agent” canrefer to any agent capable of promoting tissue formation, destruction,and/or targeting a specific disease state. Examples of bioactive agentscan include, but are not limited to, chemotactic agents, variousproteins (e.g., short term peptides, bone morphogenic proteins,collagen, glycoproteins, and lipoprotein), cell attachment mediators,biologically active ligands, integrin binding sequence, various growthand/or differentiation agents and fragments thereof (e.g., epidermalgrowth factor (EGF), hepatocyte growth factor (HGF), vascularendothelial growth factors (VEGF), fibroblast growth factors (e.g.,bFGF), platelet derived growth factors (PDGF), insulin-like growthfactor (e.g., IGF-I, IGF-II) and transforming growth factors (e.g.,TGF-β I-III), parathyroid hormone, parathyroid hormone related peptide,bone morphogenic proteins (e.g., BMP-2, BMP-4, BMP-6, BMP-7, BMP-12,BMP-13, BMP-14), transcription factors, such as sonic hedgehog, growthdifferentiation factors (e.g., GDF5, GDF6, GDF8), recombinant humangrowth factors (e.g., MP52 and the MP-52 variant rhGDF-5),cartilage-derived morphogenic proteins (CDMP-1, CDMP-2, CDMP-3), smallmolecules that affect the upregulation of specific growth factors,tenascin-C, hyaluronic acid, chondroitin sulfate, fibronectin, decorin,thromboelastin, thrombin-derived peptides, heparin-binding domains,heparin, heparan sulfate, polynucleotides, DNA fragments, DNA plasmids,MMPs, TIMPs, interfering RNA molecules, such as siRNAs,oligonucleotides, proteoglycans, glycoproteins, glycosaminoglycans, andDNA encoding for shRNA.

As used herein, the terms “biodegradable” and “bioresorbable” may beused interchangeably and refer to the ability of a material (e.g., anatural polymer or macromer) to be fully resorbed in vivo. “Full” canmean that no significant extracellular fragments remain. The resorptionprocess can involve elimination of the original implant material(s)through the action of body fluids, enzymes, cells, and the like.

As used herein, the term “carrier material” can refer to a materialcapable of transporting, releasing, and/or complexing at least onebioactive agent.

As used herein, the term “cartilage” refers to a specialized type ofdense connective tissue consisting of cells embedded in a matrix. Thereare several kinds of cartilage. Translucent cartilage having ahomogeneous matrix containing collagenous fibers is found in articularcartilage, in costal cartilages, in the septum of the nose, in larynxand trachea. Articular cartilage is hyaline cartilage covering thearticular surfaces of bones. Auricular cartilage is cartilage derivedfrom the auricle of the ear. Costal cartilage connects the true ribs andthe sternum. Fibrous cartilage contains collagen fibers. Yellowcartilage is a network of elastic fibers holding cartilage cells, whichis primarily found in the epiglottis, the external ear, and the auditorytube. Cartilage is tissue made up of extracellular matrix primarilycomprised of the organic compounds collagen, hyaluronic acid (aproteoglycan), and chondrocyte cells, which are responsible forcartilage production. Collagen, hyaluronic acid, and water entrappedwithin these organic matrix elements yield the unique elastic propertiesand strength of cartilage.

As used herein, the term “chondrogenic cell” refers to any cell which,when exposed to appropriate stimuli, may differentiate and/or becomecapable of producing and secreting components characteristic ofcartilage tissue.

As used herein, the term “function and/or characteristic of a cell” canrefer to the modulation, growth, and/or proliferation of at least onecell, such as a progenitor cell and/or differentiated cell, themodulation of the state of differentiation of at least one cell, and/orthe induction of a pathway in at least one cell, which directs the cellto grow, proliferate, and/or differentiate along a desired pathway,e.g., leading to a desired cell phenotype, cell migration, angiogenesis,apoptosis, etc.

As used herein, the term “macromer” can refer to any natural polymer oroligomer.

As used herein, the term “polynucleotide” can refer to oligonucleotides,nucleotides, or to a fragment of any of these, to DNA or RNA (e.g.,mRNA, rRNA, siRNA, miRNA, tRNA) of genomic or synthetic origin which maybe single-stranded or double-stranded and may represent a sense orantisense strand, to peptide nucleic acids, or to any DNA-like orRNA-like material, natural or synthetic in origin, including, e.g.,iRNA, ribonucleoproteins (e.g., iRNPs). The term can also encompassnucleic acids (i.e., oligonucleotides) containing known analogues ofnatural nucleotides, as well as nucleic acid-like structures withsynthetic backbones.

As used herein, the term “polypeptide” can refer to an oligopeptide,peptide, polypeptide, or protein sequence, or to a fragment, portion, orsubunit of any of these, and to naturally occurring or syntheticmolecules. The term “polypeptide” can also include amino acids joined toeach other by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain any type of modified amino acids. The term“polypeptide” can also include peptides and polypeptide fragments,motifs and the like, glycosylated polypeptides, and all “mimetic” and“peptidomimetic” polypeptide forms.

As used herein, the term “cell” can refer to any progenitor cell, suchas totipotent stem cells, pluripotent stem cells, and multipotent stemcells, as well as any of their lineage descendant cells, including moredifferentiated cells. The terms “stem cell” and “progenitor cell” areused interchangeably herein. The cells can derive from embryonic, fetal,or adult tissues. Examples of progenitor cells can include totipotentstem cells, multipotent stem cells, mesenchymal stem cells (MSCs),hematopoietic stem cells, neuronal stem cells, hematopoietic stem cells,pancreatic stem cells, cardiac stem cells, embryonic stem cells,embryonic germ cells, neural crest stem cells, kidney stem cells,hepatic stem cells, lung stem cells, hemangioblast cells, andendothelial progenitor cells. Additional exemplary progenitor cells caninclude dedifferentiated chondrogenic cells, chondrogenic cells, cordblood stem cells, multi-potent adult progenitor cells, myogenic cells,osteogenic cells, tendogenic cells, ligamentogenic cells, adipogeniccells, and dermatogenic cells.

As used herein, the term “mature chondrocyte” refers to a differentiatedcell involved in cartilage formation and repair. Mature chondrocytes caninclude cells that are capable of expressing biochemical markerscharacteristic of mature chondrocytes, including, but not limited to,collagen type II, chondroitin sulfate, keratin sulfate, andcharacteristic morphologic markers including, but not limited to,rounded morphology observed in culture and in vitro generation of tissueor matrices with properties of cartilage.

As used herein, the term “immature chondrocyte” refers to any cell typecapable of developing into a mature chondrocyte, such as adifferentiated or undifferentiated chondrocyte as well as mesenchymalstem cells that can potentially differentiate into a chondrocyteImmature chondrocytes can include cells that are capable of expressingbiochemical and cellular markers characteristic of immaturechondrocytes, including, but not limited to, type I collagen, cathepsinB, modifications of the cytoskeleton, and formation of abundantsecretory vesicles.

As used herein, the term “tracheal cartilage defect” refers to anytracheal defect of, or injury to, the trachea. Tracheal cartilagedefects may be caused by a variety of factors including, but not limitedto, stenosis caused by implanted prosthetic devices, penetrating orblunt trauma, and tumors. Additionally, tracheal cartilage defects maybe caused by congenital defects ranging from the complete absence of thetrachea to an incomplete or malformed trachea.

As used herein, the term “subject” can refer to any animal, including,but not limited to, humans and non-human animals (e.g., rodents,arthropods, insects, fish (e.g., zebrafish)), non-human primates,ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines,canines, felines, aves, etc.), which is to be the recipient of aparticular treatment. Typically, the terms “patient” and “subject” areused interchangeably herein in reference to a human subject.

As used herein, the terms “inhibit,” “silencing,” and “attenuating” canrefer to a measurable reduction in expression of a target mRNA (or thecorresponding polypeptide or protein) as compared with the expression ofthe target mRNA (or the corresponding polypeptide or protein) in theabsence of an interfering RNA molecule of the present invention. Thereduction in expression of the target mRNA (or the correspondingpolypeptide or protein) is commonly referred to as “knock-down” and isreported relative to levels present following administration orexpression of a non-targeting control RNA.

As used herein, the term “aggregate” can refer to a group or clustercomprising at least two or more cells (e.g., progenitor and/ordifferentiated cells).

As used herein, the term “population” can refer to a collection ofcells, such as a collection of progenitor and/or differentiated cells.

As used herein, the term “differentiated” as it relates to the cells ofthe present invention can refer to cells that have developed to a pointwhere they are programmed to develop into a specific type of cell and/orlineage of cells. Similarly, “non-differentiated” or “undifferentiated”as it relates to the cells of the present invention can refer toprogenitor cells, i.e., cells having the capacity to develop intovarious types of cells within a specified lineage or in differentlineages.

Embodiments described herein relate to engineered tissue constructs,such as engineered tissue rings, sheets, and disks, systems and methodsof forming modular engineered tissue constructs and to the use ofmodular engineered tissue constructs in modular tissue assembly systemsfor tissue repair and bio-artificial tissue engineering applications,such as engineered trachea constructs, engineered bone constructs,engineered vascular constructs. The engineered tissue constructs caninclude self-assembled, scaffold-free, cell aggregates with definedshapes, e.g., rings, disks, or blocks, that are formed by culturing aplurality of cells and nanoparticles and/or microparticles in wells withdefined shapes, e.g., rings, disks, or blocks, of a cell cultureapparatus or bioreactor. In some embodiments, the self-assembled, cellaggregate can include a population or plurality of cells and pluralityof nanoparticles and/or microparticles that are incorporated within thecell aggregate. The nanoparticles and/or microparticles can act as abulking agent within the cell aggregate to increase the cell aggregatesize and/or thickness. Incorporation of the nanoparticles and/ormicroparticles in the cell aggregate can also improve the mechanicalproperties of the cell aggregate formed from the cells and nanoparticlesand/or microparticles allowing the cell aggregate to be readilymanipulated and formed into engineered tissue constructs, such ascartilage rings, bone implants, and/or vascular rings. The nanoparticlesand/or microparticles can also include at least one bioactive agent thatis differentially and/or controllably released by the nanoparticlesand/or microparticles.

The engineered tissue constructs can be used in a modular tissueassembly system to engineer human tissue containing, for example,engineered cartilaginous, bone, and/or vascular segments. The tissueassembly system can permit fusion of self-assembled, scaffold-free, cellaggregates with defined shapes together to generate modular tissueconstructs with multiple types of tissues in a spatially-controlledpattern. Culture of the self-assembled, scaffold-free, cell aggregateswith defined shapes together in a hollow organ bioreactor can permitfurther modification of the constructs, including, for example,epithelialization of a surface of the construct.

Advantageously, the tissue assembly system can provide modular controlover macroscopic tissue assembly by integration of individual shapedtissue modules of different cell types, permit controlled spatial andtemporal presentation of bioactive agents to cells in the constructs,and produce constructs of various sizes and geometries usingcustomizable wells. In one example, engineered trachea constructs can beformed from engineered cartilage rings and perivascular rings and beused to rapidly fill a tracheal defect in vivo. The engineered tracheaconstructs can avoid tissue granulation, implant migration andrestenosis seen in acellular tracheal prostheses, overcome challengesregarding polymer degradation rates and byproducts presented by somescaffold-based approaches, as well as increase cell-cell interactions tohelp recapitulate de novo tissue formation by eliminating the need for ascaffold. In some embodiments, the engineered trachea constructs canutilize cells that are all of human origin and have the potential forautologous application, circumventing immune issues, potential diseasetransmission and the need for donor tissue.

FIG. 1 is a schematic illustration of an example of an engineered tissueconstruct with a defined shape in accordance with an embodiment of theapplication. The engineered tissue construct 10 has a ring-shape andincludes an engineered tissue wall 12 with an inner annular surface 14and outer annular surface 16 that extend along an axis 18 between afirst end 20 and a second end 22 of the tissue wall 12. The outerannular surface 16 defines an outer surface of the engineered tissuering 10, and the inner annular surface 14 defines an inner lumen 24 ofthe cartilage ring 10.

The outer annular surface 16 can be substantially parallel to the innerannular surface 14 to provide the tissue wall 12 and engineered tissuering 10 with a substantially uniform thickness. The thickness of thetissue wall 12 as well as the diameter and length of the tissue ring 10can be readily tailored and/or engineered for particular bioengineeringapplications. For example, the diameter of the tissue ring 10 can be atleast about 0.1 mm, about 0.5 mm, about 2 mm, about 3 mm, about 4 mm,about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm,about 11 mm, about 12 mm about 13 mm, about 14 mm, about 15 mm, about 20mm, about 30 mm, about 40 mm, about 50 mm, about 75 mm about, about 100mm or more. The length of the tissue ring can be about 0.1 mm, about 0.5mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm about13 mm, about 14 mm, about 15 mm, about 20 mm, about 30 mm, about 40 mm,about 50 mm, about 75 mm about, about 100 mm or more. The thickness ofthe tissue ring can be about 0.1 mm, about 0.5 mm, about 2 mm, about 3mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9mm, about 10 mm, about 11 mm, about 12 mm about 13 mm, about 14 mm,about 15 mm, about 20 mm, or more.

While the outer surface and the inner surface 12 and 14 are depicted asbeing substantially circular, the outer surface and inner surface 12 and14 can have other geometries including ellipsoid, toroid, frustoconical,and/or other polygonal geometries. As discussed below, the geometry ofthe inner surface and the outer surface 12 and 14 of the engineeredtissue ring 10 can be defined by the dimensions of the well used to formthe engineered tissue ring 10.

It will be appreciated that the engineered tissue construct can haveother shapes besides annular or ring shapes. These other shapes caninclude, for example, disc shapes, wedge shapes, ellipsoid shapes, andother polygonal shapes. The constructs can also include variouscavities, holes, and/or lumens. As discussed below, the specific shapeof the engineered tissue construct can be defined by the shape of thewell used to culture and form the engineered tissue construct.

The engineered tissue ring includes a self-assembled, scaffold-free highdensity cell aggregate that is formed by culturing a plurality of cellsand nanoparticles and/or microparticles in an annular well of a cellculture apparatus or bioreactor. By high density cell aggregates, it ismeant the cell aggregate has a cell density of at least about 1 × 10⁵cells/ml in cell growth medium, for example, at least about 1 × 10⁶cells/ml, at least about 1 × 10⁷ cells/ml, at least about 1 × 10⁸cells/ml, at least about 1 × 10⁹ cell/ml, or at least about 1 ×10¹⁰cell/ml in cell growth medium.

By scaffold-free, it is meant the cells are not seeded in a natural orartificial continuous polymer matrix scaffold that defines the area orvolume or at least a portion of the area or volume of the cellaggregate. A scaffold-free cell aggregate as used herein is meant todistinguish the cell aggregate from engineered tissue constructs inwhich the cells are seeded or embedded into a continuous polymer matrixor scaffold, such as a hydrogel, that encompasses the cells. Incontrast, a scaffold-free cell aggregate can include discrete or regionsof polymer or matrix materials that are intermixed with the cells andcan be in the form of nanoparticles and/or microparticles.

By self-assembled, it is meant that the cells can aggregate or assemblespontaneously or by themselves and without mechanical manipulation whilein culture into cell aggregates having defined shapes. Such assembly canbe caused by cell-cell interactions, interactions with the particles, orformation of a self-secreted extracellular matrix that can bind to orpermit the adhesion of cells in the aggregate.

In some embodiments, the self-assembled, scaffold-free, high densitycell aggregate can include a population of cells and a plurality ofnanoparticles and/or microparticles that are dispersed with the cellswithin the cell aggregate. The cell aggregate can also includeextracellular matrix material that is secreted by the cells and adheresor binds the cells and nanoparticles and/or microparticles. In someembodiments, the extracellular matrix can include collagen;proteoglycan; glycoprotein; glycosaminoglycan (GAG); as well as otherextracellular matrix proteins.

The cells used to form the cell aggregate can be autologous, xenogeneic,allogeneic, and/or syngeneic. Where the cells are not autologous, it maybe desirable to administer immunosuppressive agents in order to minimizeimmunorejection. The cells employed may be primary cells, expandedcells, or cell lines, and may be dividing or non-dividing cells. Cellsmay be expanded ex vivo prior to mixing with the nanoparticles and/ormicroparticles. For example, autologous cells can be expanded in thismanner if a sufficient number of viable cells cannot be harvested fromthe host subject. Alternatively or additionally, the cells may be piecesof tissue, including tissue that has some internal structure. The cellsmay be primary tissue explants and preparations thereof, cell lines(including transformed cells), or host cells.

In some embodiments, the cell can be an undifferentiated orsubstantially differentiated progenitor cell, such as mesenchymal stemcells, immature chondrocytes, or mature chondrocytes, human umbilicalvein, endothelial cells (hUVEC), and smooth muscle cells. In otherembodiments, the progenitor cell can be an adult stem cell, such as amesenchymal stem cell. The stem cells can be isolated from animal orhuman tissues. The stem cell used for the production of the engineeredcartilage ring can be autologous or allogeneic. In the embodimentsdescribed herein, the stem cell can isolated from, but not limited to,tendon/ligament tissue, bone morrow, adipose tissue or dental pulp. Thecell aggregate can include at least about 50%, at least about 60%, atleast about 70%, at least about 80% cells based on the total volume ofthe cell aggregate.

The nanoparticles and/or microparticles dispersed with the cells can actas a bulking agent within the cell aggregate to increase the cellaggregate size (e.g., thickness). Incorporation of the nanoparticlesand/or microparticles in the cell aggregate can also improve themechanical properties (e.g., compressive equilibrium modulus and tensilestrength) of the cell aggregate and enable more uniform extracellularmatrix deposition compared to cell aggregates without the nanoparticlesand/or microparticles. This allows the tissue rings formed from the cellaggregate to be readily manipulated and formed into tissue implants,such as trachea implants with defined architectures. The nanoparticlesand/or microparticles can also potentially enhance cell function, suchas differentiation, and/or enhance or accelerate tissue formation.

The nanoparticles and/or microparticles that are dispersed in the cellaggregate can be formed from a biocompatible and biodegradable materialthat is capable of improving properties of the cell aggregate and whichupon degradation is substantially non-toxic. The microparticles can havea diameter less than 1 mm and typically between about 1 nm and about 200µm, e.g., about 20 µm to about 100 µm. The nanoparticles and/ormicroparticles can include nanospheres, nanocapsules, microspheres, andmicrocapsules, and may have an approximately spherical geometry and beof fairly uniform size. The size and shape of the nanoparticles and/ormicroparticles dispersed in the cell aggregate can vary to adjust themechanical properties of the cell aggregate and tissue construct formedfrom the cell aggregate. In some embodiments, the nanoparticles and/ormicroparticles dispersed in the cell aggregate can have substantiallyuniform diameters; while in other embodiments, the diameters of thedispersed nanoparticles and/or microparticles can vary.

The nanoparticles and/or microparticles can include nanospheres and/ormicrospheres that have a homogeneous composition as well as nanocapsulesand/or microcapsules, which include a core composition (e.g., abioactive agent) distinct from a surrounding shell. For the purposes ofthe present invention, for the purposes of the present invention, theterms “nanosphere,” “nanoparticle,” and “nanocapsule” may be usedinterchangeably, and the terms “microsphere,” “microparticle,” and“microcapsule” may be used interchangeably.

In some embodiments, the nanoparticles and/or microparticles can beformed from a biocompatible and biodegradable polymer. Examples ofbiocompatible, biodegradable polymers include natural polymers, such ascollagen, fibrin, gelatin, glycosaminoglycans (GAG), poly (hyaluronicacid), poly(sodium alginate), alginate, hyaluronan, and agarose. Otherexamples of biocompatible, biodegradable polymers are poly(lactide)s,poly(glycolide)s, poly(lactide-co-glycolide)s, poly(lactic acid)s,poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s,polycaprolactone, polycarbonates, polyesteramides, polyanhydrides,poly(amino acids), polyorthoesters, polyacetyls, polycyanoacrylates,polyetheresters, poly(dioxanone)s, poly(alkylene alkylate)s, copolymersof polyethylene glycol and poly(lactide)s orpoly(lactide-co-glycolide)s, biodegradable polyurethanes, and blendsand/or copolymers thereof.

Still other examples of materials that may be used to form nanoparticlesand/or microparticles can include chitosan, poly(ethylene oxide), poly(lactic acid), poly(acrylic acid), poly(vinyl alcohol), poly(urethane),poly(N-isopropyl acrylamide), poly(vinyl pyrrolidone) (PVP), poly(methacrylic acid), poly(p-styrene carboxylic acid),poly(p-styrenesulfonic acid), poly(vinylsulfonicacid),poly(ethyleneimine), poly(vinylamine), poly(anhydride), poly(L-lysine),poly(L-glutamic acid), poly(gamma-glutamic acid), poly(carprolactone),polylactide, poly(ethylene), poly(propylene), poly(glycolide),poly(lactide-co-glycolide), poly(amide), poly(hydroxylacid),poly(sulfone), poly(amine), poly(saccharide), poly(HEMA),poly(anhydride), polyhydroxybutyrate (PHB), copolymers thereof, andblends thereof.

In some embodiments, the biocompatible and biodegradable polymer is abiodegradable hydrogel, such as gelatin. The biodegradable hydrogel caninclude a plurality of natural macromers that can be cross-linked usinga cross-linking agent to provide a plurality of cross-links. Varioussugar derivatives, such as glyoxal, D-ribose, or genipin can be used tocross-link the hydrogel. Other cross-linking agents, such asglutaraldehyde, can also be used. Concentrations of the crosslinkingagent as well as time and temperature used for crosslinking can bevaried to obtain the optimal results

The number or percentage of cross-links linking the macromers can bevaried to control the mechanical properties, swelling ratios, anddegradation profiles of the hydrodgel and nanoparticles and/ormicroparticles. The percentage of cross-links can be varied betweenabout 1% and about 70% by weight, and, for example, between about 20%and about 75% by weight. By increasing the percentage of cross-links,for example, the degradation rate of the biodegradable hydrogel can bedecreased. Additionally, the compressive stiffness of the biodegradablehydrogel can be increased by increasing the percentage of cross-links.Further, the swelling behavior of the biodegradable hydrogel can beincreased by decreasing the percentage of cross-links. It should also beappreciated that the macromer scaffold can be in either a hydrated orlyophilized state to enhance the addition of bioactive agents.

The nanoparticles and/or microparticles can also be modified to enhancecell function, such as differentiation, and/or enhance or acceleratetissue formation as promote cell adhesion. For example, thenanoparticles and/or microparticles can include at least one attachmentmolecule to facilitate attachment of at least one cell thereto. Theattachment molecule can include a polypeptide or small molecule, forexample, and may be chemically immobilized onto nanoparticles and/ormicroparticles to facilitate cell attachment. Examples of attachmentmolecules can include fibronectin or a portion thereof, collagen or aportion thereof, polypeptides or proteins containing a peptideattachment sequence (e.g., arginine-glycine-aspartate sequence) (orother attachment sequence), enzymatically degradable peptide linkages,cell adhesion ligands, growth factors, degradable amino acid sequences,and/or protein-sequestering peptide sequences. In one example, anattachment molecule can include a peptide having the amino acid sequenceof SEQ ID NO: 1 that is chemically immobilized onto the nanoparticlesand/or microparticles to facilitate cell attachment.

The nanoparticles and/or microparticles can also be formed frominorganic materials, such as calcium phosphate materials includingmineralite, carbonated nano-apatite, calcium phosphate based mineralite,tri-calcium phosphate, octa-calcium phosphate, calcium deficientapatite, amorphous calcium phosphate, hydroxyapatite, substituteapatite, carbonated apatite-like minerals, highly substituted carbonatedapatites or a mixture thereof. Calcium phosphate nanoparticles and/ormicroparticles can have an average particle size of between about 1 nmand about 200 µm. It will be appreciated that smaller or larger calciumphosphate nanoparticles and/or microparticles may be used. The calciumphosphate nanoparticles and/or microparticles can have a generallyspherical morphology and be of a substantially uniform size or,alternatively, may be irregular in morphology. Calcium phosphatenanoparticles and/or microparticles may be complexed with surfacemodifying agents to provide a threshold surface energy sufficient tobind material (e.g., bioactive agents) to the surface of themicroparticle without denaturing the material. Non-limiting examples ofsurface modifying agents can include basic or modified sugars, such ascellobiose, carbohydrates, carbohydrate derivatives, macromolecules withcarbohydrate-like components characterized by an abundance of —OH sidegroups and polyethylene glycol.

In some embodiments, the nanoparticles and/or microparticles can includeat least one, two, three, or more bioactive agent(s) that is capable ofmodulating a function and/or characteristic of a cell. For example, thebioactive agent may be capable of modulating a function and/orcharacteristic of a cell that is dispersed with the nanoparticles and/ormicroparticles. Alternatively or additionally, the bioactive agent maybe capable of modulating a function and/or characteristic of anendogenous cell surrounding a tissue construct formed of the cellaggregate implanted in a tissue defect.

In some embodiments, the at least one bioactive agent can include, forexample, polynucleotides and/or polypeptides encoding or comprising, forexample, transcription factors, differentiation factors, growth factors,and combinations thereof. The at least one bioactive agent can alsoinclude any agent capable of promoting cartilage, bone, or tissueformation. Examples of bioactive agents include various proteins (e.g.,short term peptides, bone morphogenic proteins, collagen, glycoproteins,and lipoprotein), cell attachment mediators, biologically activeligands, integrin binding sequence, various growth and/ordifferentiation agents and fragments thereof (e.g., EGF), HGF, VEGF,fibroblast growth factors (e.g., bFGF), PDGF, insulin-like growth factor(e.g., IGF-I, IGF-II) and transforming growth factors (e.g., TGF-βI-III), parathyroid hormone, parathyroid hormone related peptide, bonemorphogenic proteins (e.g., BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-13,BMP-14), sonic hedgehog, growth differentiation factors (e.g., GDF5,GDF6, GDF8), recombinant human growth factors (e.g., MP-52 and the MP-52variant rhGDF-5), cartilage-derived morphogenic proteins (CDMP-1,CDMP-2, CDMP-3), small molecules that affect the upregulation ofspecific growth factors, tenascin-C, hyaluronic acid, chondroitinsulfate, fibronectin, decorin, thromboelastin, thrombin-derivedpeptides, heparin-binding domains, heparin, heparin sulfate,polynucleotides, DNA fragments, DNA plasmids, MMPs, TIMPs, interferingRNA molecules, such as siRNAs, miRNAs, DNA encoding for an shRNA ofinterest, oligonucleotides, proteoglycans, glycoproteins, andglycosaminoglycans.

It will be appreciated at least one or more bioactive agent can beincorporated on or within at least one microparticle. The at least onemicroparticle can differentially or controllably release the at leastone bioactive agent or be taken up (e.g., via endocytosis) by at leastone cell to modulate the function and/or characteristic of the cell,such as to promote cartilage formation. The at least one bioactive agentmay be at least partially coated on the surface of the at least onemicroparticle. Alternatively, the at least one bioactive agent may bedispersed, incorporated, and/or impregnated within the microparticle.For example, a bioactive agent comprising a DNA plasmid (e.g., a plasmidencoding BMP-2) can be coated onto the surface of the microparticle.After forming the nanoparticles and/or microparticles with the bioactiveagent, the nanoparticles and/or microparticles can be coated with DNA orprotein to prevent nanoparticle aggregation and/or promote cellularuptake. It will be appreciated that one or more of the same or differentbioactive agents can be incorporated on or within the at least onenanoparticles and/or microparticles.

In some embodiments, a bioactive agent can comprise an interfering RNAor miRNA molecule incorporated on or within at least one microparticledispersed on or within the cell aggregate. The interfering RNA or miRNAmolecule can include any RNA molecule that is capable of silencing anmRNA and thereby reducing or inhibiting expression of a polypeptideencoded by the target mRNA. Alternatively, the interfering RNA moleculecan include a DNA molecule encoding for a shRNA of interest. Forexample, the interfering RNA molecule can comprise a short interferingRNA (siRNA) or microRNA molecule capable of silencing a target mRNA thatencodes any one or combination of the polypeptides or proteins describedabove. The at least one microparticle can differentially or controllablyrelease the at least one interfering RNA molecule or be taken up (e.g.,via endocytosis) by at least one cell to modulate a function and/orcharacteristic of the cell.

The type, distribution, size, and/or crosslinking of the nanoparticlesand/or microparticles can also be modified or configured todifferentially, controllably, spatially, and/or temporally release atleast one bioactive agent in the cell aggregate. In some embodiments,individual nanoparticles and/or microparticles can be formed ofdifferent materials or components, such as different polymers havingdifferent molecular weights or cross-linking. Moreover, thenanoparticles and/or microparticles can be formed into particular shapesor form to facilitate release of one or more bioactive agents accordingto a specific temoral release profile. Alternatively, one or morematerials or agents can be added to the nanoparticles and/ormicroparticles to facilitate differential and/or controlled release ofone or more bioactive agents according to a temporal release profile.For example, during formation of the nanoparticles and/ormicroparticles, the concentration of bioactive molecules incorporatedinto the nanoparticles and/or microparticles can be increased ordecreased to increase or decrease the concentration of the bioactivemolecules upon release from the nanoparticles and/or microparticles.

In some embodiments, the cell aggregate can include a plurality of firstnanoparticles and/or microparticles that can include or release one ormore first bioactive agent(s) and a plurality of second nanoparticlesand/or microparticles that can include or release one or more secondbioactive agent(s). The one or more first bioactive agents and the oneor more second bioactive agents may comprise the same or differentagents. The one or more first bioactive agents and the one or moresecond bioactive agents can be differentially, sequentially, and/orcontrollably released from the first nanoparticles and/or microparticlesand second nanoparticles and/or microparticles to modulate a differentfunction and/or characteristic of a cell. It will be appreciated thatthe one or more first bioactive agents can have a release profile thatis the same or different from the release profile of the one or moresecond bioactive agents from the first nanoparticles and/ormicroparticles and the second nanoparticles and/or microparticles.Additionally, it will be appreciated that the first nanoparticles and/ormicroparticles can degrade or diffuse before the degradation ordiffusion of the second nanoparticles and/or microparticles or allow foran increased rate of release or diffusion of the one or more firstbioactive agents compared to the release of the one or more secondbioactive agents. The first and second nanoparticles and/ormicroparticles may be dispersed uniformly on or within the cellaggregate or, alternatively, dispersed such that different densities ofthe first nanoparticles and/or microparticles and second nanoparticlesand/or microparticles are localized on or within different portions ofthe cell aggregate.

In some embodiments, the self-assembled, scaffold-free cell aggregatecan be formed by combining the nanoparticles and/or microparticles withthe cells and then suspending the cells and the nanoparticles and/ormicroparticles in a culture medium. The nanoparticles and/ormicroparticles can be formed, for example, from a hydrogel, such asgelatin, that is cross-linked with a cross-linking agent, (e.g.,genipin). In some instances, the nanoparticles and/or microparticles canhave a diameter of about 20 um to about 100 um and a degree ofcrosslinking of about 20% to about 70%. The nanoparticles and/ormicroparticles can also include a growth factor, such as TGFB1, that canbe loaded in the nanoparticles and/or microparticles and controllablyreleased from the nanoparticles and/or microparticles. Cell aggregatesincorporated with fast degrading nanoparticles and/or microparticlescontaining TGF-β1 produced significantly more GAG and GAG per DNA.

FIG. 2 is a schematic illustration of a method 100 of forming anengineered tissue construct that includes at least one self-assembledcell aggregate. In the method, at step 102, a population of cells can beisolated and expanded.

The cells can include any totipotent stem cell, pluripotent stem cell,or multipotent stem cell, and/or differentiated cell. Progenitor cellscan include autologous cells; however, it will be appreciated thatxenogeneic, allogeneic, or syngeneic cells may also be used. Theprogenitor cells employed may be primary cells, expanded cells or celllines, and may be dividing or non-dividing cells. The cells can bederived from any desired source. For example, the cells may be derivedfrom primary tissue explants and preparations thereof, cell lines(including transformed cells) that have been passaged once (P1), twice(P2), or even more times, or host cells (e.g., human hosts). Any knownmethod may be employed to harvest cells for use in the presentinvention. For example, mesenchymal stem cells, which can differentiateinto a variety of mesenchymal or connective tissues (e.g., adiposetissue, osseous tissue, cartilaginous tissue, elastic tissue, andfibrous connective tissues), can be isolated according to the techniquesdisclosed in U.S. Pat. No. 5,486,359 to Caplan et al. and U.S. Pat. No.5,226,914 to Caplan et al., the entireties of which are herebyincorporated by reference. In one example, the population of cells cancomprise a population of human mesenchymal stem cells.

The cells used to form the cell aggregate can include a mixture ofdifferent populations of cells or different phenotypes of cells tomodulate the properties of the engineered tissue ring. For example, thecell aggregate that forms the cartilage ring can include mixture ofchondrogenic cells, such as mesenchymal stem cells, vascular progenitorcells, such as human umbilical vein endothelial cells, and/or smoothmuscle cells.

In one example, the population of cells can comprise a population ofchondrogenic cells, such as human mesenchymal stem cells. Chondrogeniccells may be isolated directly from pre-existing cartilage tissue suchas hyaline cartilage, elastic cartilage, or fibrocartilage. Morespecifically, chondrogenic cells may be isolated from articularcartilage (from either weight-bearing or non-weight-bearing joints),costal cartilage, nasal cartilage, auricular cartilage, trachealcartilage, epiglottic cartilage, thyroid cartilage, arytenoid cartilage,and/or cricoid cartilage. Alternatively, chondrogenic cells may beisolated from bone marrow or an established cell line.

Chondrogenic cells may be allogeneic, autologous, or a combinationthereof, and may be obtained from various biological sources. Biologicalsources may include, for example, both human and non-human organisms.Non-human organisms contemplated by the present invention includeprimates, livestock animals (e.g., sheep, pigs, cows, horses, donkeys),laboratory test animals (e.g., mice, hamsters, rabbits, rats, guineapigs), domestic companion animals (e.g., dogs, cats), birds (e.g.,chicken, geese, ducks, and other poultry birds, game birds, emus,ostriches), captive wild or tamed animals (e.g., foxes, kangaroos,dingoes), reptiles and fish.

After obtaining a tissue biopsy of auricular cartilage, for example, thechondrogenic cells may be released by contacting the tissue biopsy withat least one agent capable of dissociating the chondrogenic cells.Examples of agents that can be used include trypsin and collagenaseenzymes. For example, a tissue biopsy may be sequentially digested inabout 0.25% trypsin/EDTA for about 30 minutes, about 0.1% testicularhyaluronidase for about 15 minutes, and about 0.1% collagenase type IIfor about 24 hours. The digestion may be carried out at about 37° C. inabout a 20 ml volume. Any undigested tissue and/or debris can be removedby filtering the cell suspension using a Nitex 70 µm sterile filterfollowed by centrifugation. The viability of the cells can be assessedby Trypan Blue dye exclusion test. By digesting the tissue biopsy, apopulation of chondrogenic cells comprising mature chondrocytes,immature chondrocytes, or a combination thereof, may be successfullyisolated from the tissue biopsy.

The isolated population of cells may next be expanded in a conditionedgrowth media effective to promote expansion of the cells. For example,once the chondrogenic cells have been isolated from the tissue biopsy,they may be proliferated ex vivo in monolayer culture using conventionaltechniques well known in the art. Briefly, the chondrogenic cells may bepassaged after the cells have proliferated to such a density that theycontact one another on the surface of a cell culture plate. During thepassaging step, the cells may be released from the substratum. This maybe performed by routinely pouring a solution containing a proteolyticenzyme, such as trypsin, onto the monolayer. The proteolytic enzymehydrolyzes proteins which anchor the cells on the substratum and, as aresult, the cells may be released from the surface of the substratum.

After isolation and expansion of the cells, the cells can be provided ina culture medium and mixed with nanoparticles and/or microparticles. Insome embodiments, the culture medium can also include bioactive agentsthat promote tissue formation. The nanoparticles and/or microparticlesmay be dispersed with cells in the suspension in a substantially uniformmanner. The culture medium can promote self-assembly of cell aggregatescomprising the cells and nanoparticles and/or microparticles. Inexample, the culture medium can include chemically defined basal pelletmedium (BPM) and an amount of TGF-β1 effective to stimulate cell growthand aggregation.

At step 104, the suspension can then be provided in a well, vessel,and/or chamber of a culture apparatus with a defined shape, geometry,and/or architecture. The shape of the well can define the shape of theself-assembled cell aggregate and engineered tissue construct. In oneexample, the well of the culture apparatus can have an annular shape andinclude an annular post. An outer surface of the annular post can beused to define an inner surface of a tissue ring so formed. The well ofthe culture apparatus can be formed from a biocompatible material, suchas agarose, that promotes self-assembly of the cell aggregates. In oneexample, the agarose well can be formed by molding agarose with negativemolds of machined or 3-D printed polydimethylsiloxane (PDMS).

The density at which the cells are seeded into the wells of the culturevessel can be, for example, about 1 × 10⁵ cells/mL to about 100 × 10⁶cells/mL.

The cells and nanoparticles and/or microparticles can be cultured at atemperature and atmosphere effective to promote formation of aself-assembled cell aggregate that has a shape defined by the well. Forexample, the cells may be cultured at a temperature of about 37° C. inan atmosphere of about 5% carbon dioxide at an about 90% to about 95%humidity. The oxygen percentage can be varied from about 1% to about21%. Typically, the cells can be cultured for about 1 day to about 3 ormore weeks.

Following self-assembly of the cell aggregate, at step 106, one or moreof the self assembled cell aggregates can be transferred from the wellonto a support. In one example, where the self-assembled cell aggregateis in the shape of a ring, the ring-shaped self-assembled cell aggregatecan be transferred onto a cylindrical support or tube such that thesupport extends through a lumen of the ring-shaped cell aggregate(s).The support can be made of a biocompatible material, such as siliconeand have a diameter and shape substantially the same or similar to thediameter of the lumen of the ring-shaped cell aggregate(s).

At step 108, the cell aggregate(s) positioned on the support can then beprovided in a cell differentiation medium, such as a chondrogenicinduction medium, in a cell culture vessel and cultured under conditionsdesigned to promote cell differentiation, e.g., cartilage formation. Thecells can be cultured at about 37° C. in about a 5% carbon dioxideatmosphere at about 90% to about 95% humidity. The oxygen percentage canbe varied from about 1% to about 21%. Cell differentiation medium, canbe changed daily or as needed and/or replaced with other cell culturemedium, such as osteogenic induction to promote bone formation. Othercell culture mediums can also be used, such as angiogenic medium orvasculogenic medium. The cell aggregate(s) can be cultured for aduration of time effective to promote tissue formation, for example,from about 1 week to about 4 or more weeks.

During culturing, bioactive agent(s), such as TGF-β1 and/or BMP-2, canbe released from the nanoparticles and/or microparticles via diffusionand/or as the nanoparticles and/or microparticles begin to degrade.Controlled release of the bioactive agent from the particles may bedependent on the size and composition of the nanoparticles and/ormicroparticles, as well as the composition of the medium in which theaggregate is immersed. For example, the release rate of the bioactiveagent(s) can be selectively controlled by changing the degree or percentof crosslinking of the polymers used to form the nanoparticles and/ormicroparticles, the size of the nanoparticles and/or microparticles, andthe amount of bioactive agent that is loaded into the nanoparticlesand/or microparticles.

It will be appreciated that other bioactive agents can also be added tothe medium to enhance or stimulate cell growth. Examples of otherbioactive agents include growth factors, such as transforming growthfactor-β (TGF-β) (e.g., TGF-β1 or TGF-β3), platelet-derived growthfactor, insulin-like growth factor, acid fibroblast growth factor, basicfibroblast growth factor, epidermal growth factor, hepatocytic growthfactor, keratinocyte growth factor, and bone morphogenic protein. Itwill also be appreciated that other agents, such as cytokines, hormones(e.g., parathyroid hormone, parathyroid hormone-related protein,hydrocortisone, thyroxine, insulin), fatty acids (e.g., Omega-3 fattyacids such as α18:3 linolenate), and/or vitamins (e.g., vitamin D), mayalso be added or removed from the serum-free medium to promote cellgrowth. Additionally, the engineered constructs can be mechanicallystimulated to enhance or stimulate cell growth.

The inclusion of the nanoparticles and/or microparticles in the cellaggregate can allow for substantially more uniform spatial delivery ofthe bioactive agent throughout the interior of the aggregate. Thesubstantially uniform distribution of the nanoparticles and/ormicroparticles and relatively uniform release of the bioactive agent inthe cell aggregate is advantageous for several reasons, including, butnot limited to: (1) rapidly inducing uniform cell differentiation; (2)providing control over the spatial and temporal presentation ofbioactive agents; (3) allowing for the use of lower concentrations ofbioactive agents as compared to systems employing exogenously-suppliedgrowth factors; (4) enhancing the spatial distribution of extracellularmatrix that is formed between the cells; and (5) enhancing the amount ofextracellular matrix produced in the cell aggregate. These enhancedproperties allows and/or provides for the formation of larger, moremechanically robust tissue constructs.

It will be appreciated that the cell aggregate can further include othernanoparticles and/or microparticles, such as second, third, fourth, ormore nanoparticles that include other (e.g., second, third, fourth, ormore) bioactive agents. The other bioactive agents may be the same ordifferent type of agent (described above). The other nanoparticlesand/or microparticles can differentially, sequentially, and/orcontrollably release the different bioactive agents to modulate the sameor different function and/or characteristics of at least one cell in theaggregate. The bioactive agents can have the same or different releaseprofiles from the first nanoparticles and/or microparticles.

As a result of culturing the cell aggregates with the nanoparticlesand/or microparticles, a mechanically robust engineered tissue constructwith a defined shape can be formed that can be readily shaped,transferred, and/or manipulated to form the tissue construct. In oneexample, an engineered tissue ring formed from mesenchymal stem cellscan have a glycosaminoglycan (GAG) content that can be substantiallyequal or similar to the GAG content of native cartilage.

In some embodiments, multiple self-assembled cell aggregates can befused together to form a modular engineered tissue construct. Forexample, multiple self-assembled cell aggregates having substantiallysame composition can be positioned or stacked on a support such thatportions of the self-assembled cell-aggregates abut one another on thesupport. The stacked self-assembled cell-aggregates can be cultured inthe culture medium and upon culturing can fuse together to form acontinuous substantially homogenous modular engineered tissue construct.After culturing, the continuous substantially homogenous modularengineered tissue construct can be removed from the culture vessel.

In one example, as shown in FIG. 4 , multiple ring-shaped cellaggregates having similar shapes formed in an annular well can bepositioned or stacked on a support such that adjacent ring-shapedcell-aggregates abut one another on the support. The stacked ring-shapedcell-aggregates can be cultured in a chondrogenic medium and uponculturing can fuse together to form a continuous substantiallyhomogenous engineered tissue tube.

In other embodiments, a heterogenous modular engineered tissue constructcan be formed that includes defined regions or portions (e.g., rings) ofdiffering or similar cell aggregate materials. The differing regions orportions of the heterogenous modular engineered tissue construct can beprovided or formed with or without nanoparticles and/or microparticlesand can have similar or different properties to vary the properties ofthe tissue construct for particular tissue engineering applications.

In some embodiments, a heterogenous modular tissue construct can beformed by fusing self-assembled cell aggregates formed from differentmixtures of cells with or without nanoparticles and/or microparticles.For instance, a first mixture of cells with or without nanopaticlesand/or microparticles can be seeded into wells of a culture chamber toform first self-assembled cell aggregates. A second mixture of cellswith or without nanoparticles and/or microparticles different than thefirst mixture can be seeded in the same or separate wells of a culturechamber to form second self-assembled cell aggregates. The first mixtureof nanopaticles and/or microparticles and cells can include differenttype, concentration, amount, and/or distribution, of cells,nanoparticles and/or microparticles and/or potentially bioactive agentsas the second mixture of nanopaticles and/or microparticles and cells tovary the compositions and properties of the first self-assembled cellaggregates and the second self-assembled cell aggregates.

The first self-assembled cell aggregates and the second self-assembledcell aggregates can be transferred from the wells onto a support suchthat portions of the self-assembled cell-aggregates abut one another onthe support. In some embodiments, one or more first self-assembled cellaggregates can be alternated with one more second self-assembled cellaggregates on the support such that different self-assembled aggregatesare in contact with each other. The first and second self-assembled cellaggregate(s) positioned on the support can then be provided in a cellculture medium in a cell culture vessel and cultured under conditionsdesigned to fuse the separate aggregates and form a modular engineeredtissue construct.

It will be appreciated that the heterogenous modular engineered tissueconstruct can be formed by fusing more than two different types ofself-assembled cell aggregates. Each of the different self-assembledcell aggregates can include differing mixtures of cells with or withoutnanopaticles and/or microparticles and be fused together in anycombination, e.g., alternating in series, etc. Additionally, each of thedifferent self-assembled cell aggregates can have different shapes sothat engineered tissue constructs formed by fusing the differentself-assembled cell aggregates can be provided with complex shapes andgeometries.

In one example, as shown in FIGS. 3 , first ring-shaped cell aggregatesformed form a mixture of human mesenchymal stem cells and TGF-β1 loadedmicroparticles and/or nanoparticles and second ring-shaped cellaggregates formed from smooth muscle cells can be stacked on a supportto provide alternating rings of the first ring-shaped cells aggregatesand the second ring-shaped aggregates. The first and second ring-shapedcell aggregate(s) positioned on the support can then be provided in acell culture medium in a cell culture vessel and cultured underconditions designed to fuse the rings and form multi-tissue type hMSCand smooth muscle cell tubes with both cartilaginous andnon-cartilaginous portions.

In other examples, first ring-shaped cell aggregates formed form amixture of human mesenchymal stem cells and TGF-β1 and/or BMP-2 loadedmicroparticles and/or nanoparticles and second ring-shaped cellaggregates formed from a mixture of human mesenchymal stem cells andhuman umbilical vein endothelial cells (e.g., a 1:1 mixture of MSCs tohUVEC) can be stacked on a support to provide alternating rings of thefirst ring-shaped cells aggregates and the second ring-shapedaggregates. The first and second ring-shaped cell aggregate(s)positioned on the support can then be provided in a cell culture mediumin a cell culture vessel and cultured under conditions designed to fusethe rings and form multi-tissue type hMSC and hUVEC tubes with bothcartilaginous and prevascular portions.

Optionally, a homogenous or heterogeneous modular engineered tissueconstruct formed by the method described herein can be further modifiedby seeding cells onto or within the homogenous or heterogeneous modularengineered tissue construct. In one example, where the homogenous orheterogeneous modular engineered tissue construct is in the form of aring or tube used for a trachea implant, respiratory epithelial cellscan be seeded on an inner surface of a lumen of the ring or tube to forma bilayer cell tube or ring. It will be appreciated that other cells orcell types can be seeded onto or within the homogenous or heterogeneousmodular engineered tissue construct to modulate the properties of thehomogenous or heterogeneous modular engineered tissue construct and forma heterogenous or multilayer structure.

The homogenous or heterogeneous modular engineered tissue constructproduced by the method described herein can find use in a variety ofapplications. One example of such an application can include forming awhole or partial portion of a trachea implant to treat a tracheal defectin a subject. In some embodiments, the tracheal implant can include aheterogeneous cartilage tube with alternating fused cartilaginous andnoncartilaginous portions and an inner lumen in which is seededepithelial cells to provide an epithelial lined implant. Depending uponthe clinical needs of the subject, homogenous or heterogeneous cartilagering or tube produced by the methods described herein may be used toform a whole trachea or only a portion of a whole trachea. For example,a tracheal implant may be formed by first obtaining a homogenous orheterogeneous modular engineered tissue ring or tube that includecartilage rings and/or vascular rings and/or prevascular rings, with orwithout epithelial cell lining. The tracheal implant may be optimallysized to suit the needs of the subject. The implant may be used torepair a tracheal cartilage defect as described in greater detail below.

Repair of a tracheal cartilage defect may begin by first identifying thedefect. Tracheal cartilage defects may be readily identifiable byvisually identifying the defects during open surgery of the trachea or,alternatively, by using computer aided tomography, X-ray examination,magnetic resonance imaging, analysis of serum markers, or by any otherprocedures known in the art.

Once the tracheal cartilage defect has been identified, anappropriately-sized tracheal implant may be selected. For example, thetracheal implant may have a size and shape so that when the trachealimplant is implanted, the edges of the tracheal implant directly contactthe edges of native cartilage tissue. The tracheal implant may be fixedin place by, for example, surgically fixing the implant withbioresorbable sutures. Additionally or optionally, the tracheal implantmay be fixed in place by applying a bioadhesive to the regioninterfacing the tracheal implant and the tracheal cartilage defect.Examples of suitable bioadhesives include fibrin-thrombin glues andsynthetic bioadhesives similar to those disclosed in U.S. Pat. No.5,197,973.

The cartilage tissue defect may comprise a stenotic portion of thetrachea, such as two of the cartilages comprising the trachea, caused byprolonged placement of a tracheal T-tube. To repair the trachealcartilage defect, the stenotic portion may first be surgically excised.Next, a tracheal implant may be formed having a size and shapecomplementary to the size and shape of the excised stenotic portion. Thetracheal implant may then be surgically fixed in place of the excisedstenotic portion by an end-to-end anastomosis. After the trachealimplant has been suitably fixed in place, the surgical procedure may becompleted and the tracheal implant permitted to integrate into thenative cartilage tissue.

In an alternative example, the tracheal cartilage defect may comprise acongenital defect, such as a missing trachea, in a pediatric subject. Atracheal implant comprising a whole trachea may be prepared and thensurgically implanted into the subject by an end-to-end anastomosis.After the tracheal implant has been suitably fixed in place, thesurgical procedure may be completed and the tracheal implant permittedto integrate into the native tissue. By providing the subject with awhole tracheal implant, the tracheal implant may integrate into thenative tissue and grow along with the subject, thus removing the need toperform additional surgeries as the subject ages.

It will be appreciated that the homogenous or heterogeneous modularengineered tissue construct produced by the methods described herein canalso be used to form tissue constructs other than engineered trachea.Such tissue constructs can include, for example, vasculature implantsfor vasculature repair, bone implants that potentially include multiplelayers or modular structures, tubular tissues or organs, such as theesophagus, small intestines, urethra, vagina, and muscular tubes (e.g.,cardiac and skeletal muscle), other organs or tissue, or other implantsused to repair tissue or cartilage defects. Tissue defects in thecontext of the present invention should also be understood to comprisethose conditions where surgical repair of tissue is required, such ascosmetic surgery (e.g., nose, ear). Thus, tissue defects can occuranywhere in the body where tissue formation is disrupted, where tissueis damaged or non-existent due to a genetic defect, where tissue isimportant for the structure or functioning of an organ (e.g., structuressuch as menisci, the ear, the nose, the larynx, the trachea, thebronchi, structures of the heart valves, part of the costae,synchondroses, enthuses, etc.), and/or where tissue is removed due tocancer, for example. For such applications, the homogenous orheterogenous modular engineered tissue construct can be shaped, molded,or configured into a variety of configurations.

In still other embodiments, the homogenous or heterogeneous modularengineered tissue construct produced by the methods described herein canbe combined with or adhered to other tissue constructs to form aheterogeneous tissue constructs. For example, a modular engineeredtissue ring or tube produced by the methods described herein can beprovided on, combined with or adhered to demineralized bone matrix toprovide and osteochondral tissue construct. The tissue construct can bereadily implanted and integrated osteochondral defect.

In other embodiments, the DNA or cells in the homogenous orheterogeneous modular engineered tissue construct produced by themethods described herein can be removed or lysed to provide an acellulartissue construct that includes the extracellular matrix so formed and,potentially, the partially or completely degraded nanoparticles and/ormicroparticles. Removal may be achieved by, for example, detergenttreatment, (e.g., SDS treatment) treatment with DNase and RNase, and/orfreeze/thaw cycles. The acellular tissue construct can then be usedalone for tissue engineering application or in combination with othercell types or growth factors for the promotion of tissue repair. Theacellular tissue construct can be used as an acellular biomaterial fortissue engineering application similar to the above afterdecellularization. When used alone, the acellular tissue can be used toprevent or repair tissue defects, enhance host cell attachment,infiltration, differentiation, extension, and proliferation. Theacellular tissue construct as a decellularized product can be usedtogether with other known bioactive agents and cell types for thepromotion of tissue repair.

The following examples are for the purpose of illustration only and arenot intended to limit the scope of the claims, which are appendedhereto.

Example 1

In this Example a tracheal tissue replacement strategy is demonstratedusing a bottom-up approach for production of human MSC (hMSC)-derivedcartilaginous rings and tubes through employment of custom designedculture wells and an assembly system. This technology is then used totest the hypothesis that incorporation of chondrogenic growthfactor-delivering microspheres into the ring and tube-shaped high-celldensity constructs enhances chondrogenesis with regard to mechanicalproperties and matrix production and distribution to provide functionaltracheal patency in future clinical applications.

Methods Experimental Design

The work described here investigated the formation of engineeredcartilaginous rings and tubes in custom designed molds. hMSCs alone(“hMSC”) or with bioactive factor-releasing biopolymer microspheres(“hMSC + MS”) were seeded in annular agarose wells to form scaffold-freeself-assembled three-dimensional tissue rings. Subsequently, tissuerings were stacked in 3-ring or 6-ring conformations to fuse into tissuetubes. Chondrogenesis was induced in rings and tubes during 22 days ofin vitro culture after which constructs were harvested for analysis. Aschematic of the ring and tube formation procedure is shown in FIG. 4 .hMSC isolation and culture

hMSCs were isolated from bone marrow aspirates obtained from the CaseComprehensive Cancer Center Hematopoietic Biorepository and CellularTherapy Core under University Hospitals of Cleveland InstitutionalReview Board approval, as previously described. Briefly, bone marrowaspirates were washed with expansion media (Dulbecco’s Modified Eagle’sMediumelow glucose (DMEM-LG; Sigma-Aldrich, St. Louis, MO)) containing10% pre-screened bovine serum (Gibco Qualified FBS; Life Technologies,Carlsbad, CA). Mononuclear cells were separated using a Percoll gradient(Sigma-Aldrich), plated in expansion media and cultured in a 37° C.humidified incubator with 5% CO₂. Non-adherent cells were washed awayduring the first media change. Adherent cells received fresh expansionmedia supplemented with 10 ng/ml fibroblast growth factor-2 (FGF-2, R&DSystems, Minneapolis, MN) every 2-3 days. The hMSCs were subcultured at~90% confluence, and passage 3 cells were used in this study.

Microsphere Synthesis and Characterization

Gelatin microspheres (11.1 w/v% Type A; Sigma-Aldrich) were synthesizedin a water-in-oil emulsion, as previously described, with slightmodifications. Microspheres were crosslinked with 1 w/v% genipin for 3 h(Wako Chemicals USA Inc., Richmond, VA), washed with deionized H₂O,lyophilized and rehydrated with Dulbecco’s Phosphate Buffered Saline(PBS; HyClone Laboratories, Logan, UT) containing 400 ng TGF-b1(PeproTech, Rocky Hill, NJ) per mg microspheres. Light microscopy imagesof hydrated, crosslinked microspheres (N = 268) were acquired on a TMSmicroscope (Nikon, Tokyo, Japan) with a Coolpix 995 camera (Nikon) todetermine microsphere diameters, which weremeasured using NIH Image Janalysis software. The degree of microsphere crosslinking was quantifiedvia a ninhydrin assay, based on a previously described protocol. Here,the ninhydrin solution was added to dry microspheres and incubated for2.5 min.

Cell Culture Well Preparation

Agarose molds for cell culture were prepared as follows. Briefly, apolycarbonate sheet (Small Parts Inc., Miramar, FL) was machined tocontain annular wells with concentric 2 mm diameter posts surrounded bya 3.75 mm wide trough. A polydimethylsiloxane (PDMS; Sylgard 184, DowComing, Midland, MI) negative mold of the polycarbonate template wascured and steam autoclaved for sterilization. Two percent w/v agarose(Denville Scientific Inc., Metuchen, NJ) in DMEM-LG was autoclaved andused to fill the PDMS mold. After cooling, the ring-shaped culture wellswere removed from the PDMS mold, moved into 6-well plates (BD, FranklinLakes, NJ) and incubated overnight in basal pellet medium (BPM)comprised of Dulbecco’s Modified Eagle’s Medium, high glucose (DMEM-HG;Sigma Aldrich), 1% ITS þ Premix (Corning Inc, Corning, NY), 10⁻⁷ Mdexamethasone (MP Biomedicals, Solon, OH), 1 mM sodium pyruvate (HyCloneLaboratories), 100 µM non-essential amino acids (Lonza Group, Basel,Switzerland), 37.5 µg/ml ascorbic acid-2-phosphate (Wako Chemicals USAInc.) and 100 µ/ml penicillin-streptomycin (Corning Inc.).

Assembly of Microsphere-Containing Tissue Rings and Tubes

Trypsinized hMSCs (400,000 cells) with or without 0.3 mg TGF-β1 ladenmicrospheres in 50 µL media were seeded in a circular fashion in eachcustom designed annular well. Microsphere-containing tissues (“hMSC +MS”) were seeded and cultured in BPM. hMSC-only groups (“hMSC”) did notcontain microspheres and were seeded and cultured in BPM supplementedwith 10 ng/ml TGF-β1. After 24 h, 3 ml of experimentalcondition-specific media were added to the agarose wells. On day 2, someof the self-assembled rings were transferred from the annular wells onto2 mm silicone tubes (Specialty Manufacturing Inc., Saginaw, MI) to form3- and 6-ring tissue tubes. Silicone tubes were sandwiched betweencustom engineered polycarbonate holders and the developing tissue tubeswere cultured horizontally in 60 mm petri dishes (BD) containing 4.8e6million cells and 9 ml of condition specific media. A schematic of thetissue ring and tube assembly processes is shown in FIG. 5 . A Galaxy S4phone camera (Samsung, Seoul, Korea) was used to capture images of thecustom culture set-up right after tissue tube assembly. Tissue rings andtubes with and without microspheres were grown in a humidified cellculture incubator at 37° C. and 5% CO₂ for 22 days with media changesevery 2 and 3 days, respectively.

Gross Morphological Assessment

On day 22 of total culture, rings (22 days of culture as rings) andtubes (2 days of culture as rings followed by 20 days of culture astubes) were harvested and photographs of all tissues were taken with aGalaxy S4 phone camera. Healthy, native rat tracheas (male NIH Nude rats14e15 weeks old (N = 4); Taconic, Hudson, NY), freshly harvested fromrats sacrificed for another study in accordance to a protocol approvedby the Institutional Animal Care and Usage Committee at Case WesternReserve University, were used for comparison.

Biochemical Analysis

Tissue rings (N = 4) and 3-ring tubes (N = 3) were digested in papainsolution (Sigma Aldrich) at 65° C. GAG and DNA contents were measuredusing dimethylmethylene blue (DMMB; Sigma-Aldrich) and PicoGreen(Invitrogen, Carlsbad, CA) assays, respectively.

Histology and Immunohistochemistry

Tissue rings and 3-ring tubes (N = 2) were fixed in 10% neutral bufferedformalin overnight, embedded in paraffin and sectioned at 5 microns.Rings were sectioned in either axial or vertical planes. Tubes weresectioned first in the axial plane and then reembedded in paraffin andsectioned in the vertical plane. Mounted tissue sections weredeparaffinized and rehydrated. Safranin O (Acros Organics) was used tostain for sulfated GAG content with a Fast Green counterstain (FisherChemical). For immunohistochemical staining, the presence of type IIcollagen was detected using anticollagen type II primary antibody (abcamab34712, Cambridge, UK) with a Fast Green counterstain. A section of thehuman knee articular cartilage and underlying subchondral bone served asa positive and negative control, respectively. Samples stained withisotype-matched IgG instead of primary antibody also served as negativecontrols. Histostain-Plus Bulk kit (Invitrogen) with aminoethylcarbazole (AEC; Invitrogen) was used to visualize the primary antibody.Images of stained tissues were acquired using an Olympus BX61VSmicroscope (Olympus, Center Valley, PA) with a Pike F-505 camera (AlliedVision Technologies, Stadtroda, Germany).

Tissue Dimension Measurements and Biomechanical Analysis Rings

Day 21 tissue engineered rings and rat tracheal sections were sent inchondrogenic media from Case Western Reserve University to WorcesterPolytechnic Institute (transit time was 3 nights and 1 night,respectively). Rings were then allowed to equilibrate for approximately2 h in a 37° C. incubator prior to mechanical testing. Tissue ring wallthickness was measured in PBS using a machine vision system (DVT Model630; DVT Corporation, Atlanta, GA). Measurements were taken in fourlocations around each ring using edge detection software (Framework2.4.6; DVT), and the average thickness was used to calculate the averagecross-sectional area. Each rat trachea was also measured in fourlocations, but using calipers due to its more uneven shape. Tissueengineered rings with and without microspheres and rat trachea sectionswere tested in uniaxial tension (ElectroPuls E1000 with a 50 N loadcell; Instron, Norwood, MA) using a modified version of a systemdescribed previously. Briefly, small stainless steel pins were bent intoan “L” shape and served as grips for individual rings (FIG. 11A inset).After applying a 5 mN tare load, engineered rings were pulled in tensionto failure at a rate of 10 mm/min. PBS was dripped on tissues duringtesting to prevent drying. From this test, the maximum load the ringscould withstand was calculated. The ultimate tensile stress (UTS) wascalculated by dividing the failure load by the cross-sectional area.Each engineered ring was approximated as a torus and each native tracheasection was approximated as a hollow cylinder. Tubes

Tissue engineered 6-ring tubes and 8 mm sections of rat trachea wereequilibrated in PBS with 0.1% protease inhibitor (Sigma-Aldrich), andtheir outer diameters were measured by applying a pre-load of 3 mN withan R Series Controller mechanical testing device (Test Resources Inc.,Shakopee, MN). Individual tubes and tracheas were tested in luminalcollapse as previously described with modifications. Each tube andtrachea was compressed by 2 mm (luminal diameter) at a rate of 0.5mm/min. The load was held for 6 min and then was removed at a rate of 60mm/ min. The load to collapse the lumen by 80% (1.6 mm) was used forcomparison between the engineered tubes and rat tracheas. This was doneto ensure that only the load required to collapse the lumen withoutcompressing the walls of the tube was analyzed. Tube outside diameterwas measured again after a 5 min no-load period. Percent luminal recoilwas calculated as the ratio of the final outer diameter/ initial outerdiameter *100. Video recordings (Galaxy S4 phone camera) were taken of arepresentative hMSC tube, hMSC þ MS tube and a section of rat trachea(after 1 freeze/thaw) compressed by a hand-held pipet to show repetitiveluminal collapse and recoil of the tubes.

Statistical Analysis

One-way ANOVA with Tukey’s post hoc tests were used to statisticallyanalyze tissue engineered constructs and native tracheas via InStat 3.06software (GraphPad Software Inc., La Jolla, CA). All values are reportedas mean ± standard deviation. Post tests were performed when p < 0.05.

Results Microsphere Characterization

Gelatin microspheres appeared blue as a result of the crosslinkingreaction with genipin. They were 26.6 ± 8.0% crosslinked, and theiraverage diameter was 67.8 ± 55.1 mm (N = 268). A representative lightmicroscopy image shows microspheres size variability (FIG. 5 ). Severalhours after seeding, hMSC and hMSC + MS rings had self-assembled aroundthe posts. After 2 days of culture, hMSC microsphere-containing ringsappeared thicker and darker due to presence of microspheres compared tohMSC-only tissues, which were opaque off-white (FIGS. 6A and C). Thesurface of hMSC þ MS rings was less smooth compared to that of hMSC-onlyrings. Both hMSC-only and hMSC þ MS rings could be handled with tweezersfor tissue tube assembly into 3-ring or 6-ring tubes (FIGS. 6B and D),but microsphere-containing rings held their toroid shape better duringtransfer from the agarose posts to the silicone tubes.

Gross Morphological Assessment

Tissues harvested after 22 days of total culture were firm and could beeasily handled. The thickness of hMSC-only rings was more irregularcompared to microsphere-containing rings, which were visually thickerand slightly blue due to residual microspheres that were not fullydegraded (FIGS. 7A and D). Stacked rings formed fused 3-ring or 6-ringtissue tubes on the 2 mm silicone tubing (FIGS. 7B-F). Rings and 3-ringtissue tubes were pink due to residual media in the tissue, while 6-ringtissue tubes and rat tracheas were rinsed in PBS before beingphotographed. Similar to the microsphere-containing rings, tubes withmicrospheres were visually thicker than hMSC-only tubes. hMSC + MS tubeswere also longer than the hMSC-only tubes. Incorporation of microspherescontributed to formation of ridged surfaces on tubes compared to smoothsurfaces on hMSC-only tubes. Rat tracheas had visibly thinner wallscompared to tissue engineered tubes (FIG. 7G).

Biochemical Analysis

Individual rings and 3-ring tubes were analyzed biochemically. Asexpected, DNA (FIG. 8A), an indirect measure of cell number, and GAG(FIG. 8B) content were significantly greater in tubes compared toindividual rings because 3 rings were used for each tube. There was nosignificant increase in GAG production per DNA (FIG. 8C) in tissuesgrown in ring compared to tube geometries. Addition of growthfactor-delivering microspheres did not significantly affect the cellnumber at the time of harvest as measured by amount of DNA. However,microspheres significantly increased total GAG and GAG production percell. GAG/DNA was greater in hMSC þ MS tissues than those without MS byfactors of 2.2 and 1.7 in rings and tubes, respectively.

Histology and Immunohistochemistry

Safranin O with a Fast Green counterstain was used to visualize thepresence and distribution of GAG in tissue engineered rings (FIGS. 9Aand C), 3-ring tubes (FIGS. 9B and D) and rat trachea (FIG. 9E). Ringsand tubes with microspheres (FIGS. 9C and D) stained more intensely forGAG compared to cell-only constructs (FIGS. 9A and B), corroborating thebiochemical analysis. hMSC + MS rings and tubes were also visuallythicker and had a more uniform GAG distribution with a smaller fibrouscapsule (stained blue/green by Fast Green) on the tissue peripherycompared to the hMSC-only tissues. The remaining gelatin microspheresthat were not fully degraded by cell-secreted enzymes were visible inthe hMSC þ MS groups (black arrows in FIGS. 9C and D). Cartilaginousportions of the rat trachea had the most intense GAG staining. Verticalcross sections of tissue engineered tubes of both compositions showedseamless ring fusion. hMSC-only tubes appeared to have lower GAG densityin the middle of the constructs. Microsphere-containing tubes maintainedridges from the individual rings that were fused together. Cartilagerings in the rat trachea were separated by noncartilaginous fibroustissue, which stained blue/green.

The presence and distribution of collagen type II were visualized viaimmunohistochemical staining (FIG. 10 ). hMSC and hMSC + MS rings andtubes both showed strong staining for type II collagen, which was moreprevalent on the interior of the constructs. However, staining wasbetter distributed in microsphere-containing tissues. Human knee tissuecontrol showed appropriate collagen type II staining of articularcartilage while not staining the subchondral bone.

Tissue Dimension Measurements and Biomechanical Analysis Rings

The walls of engineered hMSC-only and hMSC + MS cartilaginous rings weresignificantly thicker than those of native rat tracheas (FIG. 11A).Incorporation of microspheres resulted in rings that were significantlythicker than their cell-only counterparts. Uniaxial tension mechanicaltesting (FIG. 12A inset) revealed that the maximum force at failure(FIG. 12A) was similar in the engineered rings, but rat tracheal ringsrequired a significantly smaller load to rupture. However, when force atfailure was normalized to loaded area (ultimate tensile stress; FIG.11B), microsphere-containing rings (2.44 ± 0.22 mm² cross-sectionalarea) behaved similarly to the rat trachea (1.22 ± 0.16mmlong; 1.11 ±0.19mm2 cross-sectional area), while hMSC-only rings (1.26 ± 0.24 mm2cross-sectional area) exhibited significantly greater stress at failurethan the other two groups.

Tubes

Tissue engineered tubes had a significantly greater outer diameter thanthe rat tracheas (FIG. 11B). In addition, microspheres containing tubeshad a significantly greater outer diameter than hMSC tubes. In a grossbiomechanical assessment of the tissue tubes, the qualitative forcerequired to collapse the tubes with a hand-held pipet was the largestfor the hMSC þ MS tube (FIG. 13 ). Quantitative mechanical analysiscorroborated the qualitative findings. A force was applied to collapsethe tubes by 2 mm, the engineered tissues’ inner diameter (FIG. 12Cinset). The force required to collapse 80% of the lumen of engineeredhMSC + MS tubes were about 2.1-2.3 times greater than the force requiredto collapse hMSC-only tubes and similarly-sized, 8 mm length sections ofrat tracheas (FIG. 12C). Cell-only tubes required approximately the sameload to achieve luminal collapse compared to the hMSC + MS tubes. Afterthe load was removed, the outer diameters of the tubes were measuredagain and it was found that all tubes recoiled to nearly 100% of theiroriginal diameters (FIG. 12D).

This Example demonstrates the ability to form cartilaginous rings fromhuman bone marrow-derived MSCs in custom culture wells and stack therings to generate fused tissue tubes. Secondly, this work shows thatincorporation of microspheres delivering chondrogenic growth factor(i.e., TGFβ1) into the self-assembled ring- and tube-shaped constructswould enhance neocartilage formation by increasing matrix production,tissue dimensions and mechanical properties. Custom annular culturewells comprised of agarose were used to successfully engineer hMSC-onlyand microsphere containing rings. On day 2 of culture, rings could bemanipulated and stacked onto a silicone tube to fuse into tissue tubes.The two days of culture needed in this study is a much shorter time thanthe previously reported 3-4 week culture period necessary for high-celldensity chondrocyte sheets to achieve mechanical integrity required formanual manipulation. After approximately 3 weeks of culture, tubes wereeasily removed from the silicone support and exhibited seamless fusionbetween rings as observed via gross morphological and histologicalevaluation (FIGS. 7, 9 and 10 ). The homogenous fusion between 2-day-oldhigh-density hMSC-derived cartilage rings are consistent with thepreviously reported fusion of high density hMSC pellets undergoingchondrogenesis. The presence of GAG (FIGS. 8 and 9 ) and collagen II(FIG. 10 ) indicated cartilaginous tissue formation after 22 days oftotal culture. These findings confirmed that custom agarose molds can beused to engineer cartilaginous rings and that these rings can be fusedinto tissue tubes.

While there are reports describing fabrication of rabbit auricularchondrocyte-derived cartilage sheets that were rolled to fuse into atube in vitro or in vivo, our approach has advantages over thesesystems. First of all, human bone marrow MSCs used here as the cellsource for autologous cartilage tissue formation avoids the need forinvasive and potentially detrimental harvest of mature cartilage tissuesand provides the capacity for cell expansion to achieve necessarynumbers of cells capable of undergoing chondrogenesis. In addition, theuse of human cells in our system is potentially a more translatablestrategy, as approaches utilizing cells from different species mayresult in different chondrogenic outcomes compared to those with humancells, delaying or inhibiting transfer of technology to the clinicalsetting. Secondly, the hMSC-based cartilaginous rings were significantlythicker than previously reported chondrocyte-based approaches. In just 3weeks of culture, hMSC derived cartilage rings were 0.89 mm (hMSC-only)and 1.25 mm (hMSC + MS) thick compared to rabbit articularchondrocytederived cartilage, which was 235 mm thick after 8 weeks ofculture, and rabbit auricular chondrocyte-derived cartilage, which wasabout 500 mm thick after 6 weeks of culture and 553 mm thick after 8weeks of culture. To achieve wall thicknesses similar to those found inour hMSC-based rings, multiple cartilage sheets would need to be stackedor folded. Thirdly, in terms of cartilage tube fabrication, the ringassembly system does not require the binding of tissue sheets withsutures or ties to form a tubular construct. More importantly, ourring-based approach is a modular system which could prove advantageouswhen generating multi-tissue type constructs because each ring couldserve as a tissue building block. Finally, the ring-to-tube technologymay be more favorable in resisting compression in the axial plane,thereby maintaining tracheal patency in future in vivo applications,compared to sheet-to- tube technologies which may have heterogeneousmechanical properties around the circumference of the tube. While rathepatocyte cell line rings and 2-ring tubes, normal human fibroblastrings and smooth muscle cell rings and tubes have been reported, thefabrication of scaffold-free, stem cell based cartilage-like rings andtubes using a custom ring and tube assembly system has not yet beendemonstrated.

The degree of chondrogenesis was also compared between tissues developedfrom high-density hMSC ring and tube cultures containingproteolytically-degradable TGF-β1-loaded gelatin microspheres andhMSC-only tissues grown in the same geometries with exogenouslydelivered growth factor. This high-cell density culture system withbioactive microspheres has previously been shown to enhancechondrogenesis, mechanical properties and/or tissue thickness inhMSC-derived aggregate and sheet constructs. In the present work,tissues with bioactive microsphere were visually thicker (both rings andtubes) and longer (tubes) than hMSC-only constructs. Tubes created frommicrosphere containing rings maintained outer ridge morphologyreminiscent of the rings used for fusion (FIGS. 7E, F and 9D). It ispossible that even after only 2 days of culture, incorporation ofTGF-β1-loaded microspheres encouraged greater and/or more mature matrixdeposition in tissue rings, making the remodeling of ECM morechallenging during the fusion process. Another possible reason for thepresence of ridges in the hMSC + MS tubes is that incorporation ofmicrospheres led to a more uniform cartilaginous matrix distribution anda reduced fibrous capsule, which has been reported to encouragecartilage tissue fusion. This GAG-poor capsule, sometimes seen on theperiphery of high-cell density cultures, was more prevalent in hMSC-onlytissues and may be the reason for smoother surfaces found in hMSC-onlytubes (FIGS. 9A and B) compared to the GAG-rich, ribbed hMSC + MS tubes(FIGS. 9C and D).

Incorporation of growth factor-loaded microspheres enhancedchondrogenesis as detected by biochemical and histological assays andmeasurement of tissue dimensions. This finding is corroborated byprevious reports of improved cartilage formation in high-density hMSCsystems with incorporated TGF-b1-loaded gelatin microspheres. Comparedto hMSC-only rings and tubes, hMSC + MS rings and tubes produced moreGAG per DNA (FIG. 8C) and stained more intensely for GAG (FIG. 9 ) andcollagen type II (FIG. 10 ), which are all indicative of neocartilageformation. Not only was the ECM more cartilaginous, addition of growthfactor loaded microspheres led to increased tissue ring thickness andtube outer diameter (FIG. 11 ). Taken all together, the biochemical,histological and tissue dimension data supported our hypothesis thatincorporation of growth-factor-loaded microspheres into the hMSChigh-cell density rings and tubes improved chondrogenesis in theconstructs.

Upon mechanical evaluation of tissue engineered rings and tubes,incorporation of microspheres decreased ring tensile strength and didnot affect tubular luminal elasticity. Uniaxial UTS values showed thatincorporation of microspheres resulted in a lower stress at failure(FIG. 12B). Even though the load at failure was only slightly lower inhMSC + MS rings than hMSC-only rings (no significant difference; FIG.12A), the hMSC + MS rings had a significantly greater cross-sectionalarea (FIG. 11A) resulting in a significantly smaller UTS. Still, reducedUTS values were an unexpected finding since incorporation of growthfactor-laden microspheres has been shown to increase the equilibriumcompressive moduli of hMSC-derived engineered cartilage sheets. However,the residual gelatin microspheres that were not fully degraded couldhave been acting as inclusions, thereby weakening the tissues’ tensilestrength. Another potential explanation for decreased UTS is thedifferences in biochemical make-up of the ECM in the hMSC + MS comparedto hMSC-only rings. The GAG content is a dominant contributor toincreased tissue stiffness in compression and collagen content ispredominantly responsible for tensile properties in cartilage tissues.While the addition of microspheres significantly increased GAGbiochemical content as well as GAG and type II collagen staining, it ispossible that microspheres led to a greater relative increase in GAGcompared to collagen resulting in lower tensile properties in the hMSC +MS rings than the hMSC-only rings. Mechanical evaluation of tissue tubesshowed that all tissue tubes recoiled to almost the original outerdiameter, but microsphere-containing tissues required greater force tocollapse the tubes. However, microsphere containing tubes were alsoqualitatively longer than their cell-only counterparts so it isdifficult to examine the role that microspheres played on the luminalelasticity mechanics of the tissue tubes.

Tissue generation was influenced by culture in the custom wells andassembly system in the ring and tube geometries. Three-ring tubes hadsignificantly greater DNA and GAG content than individual rings,although these increases were slightly less than the 3- foldproportional increases that would be expected. However, GAG productionper cell was not significantly different between ring and tubegeometries. Unexpectedly, with and without microspheres, both geometriesled to approximately two-fold greater GAG/DNA production compared tohigh-cell density sheets grown on cell culture inserts using samepassage hMSCs from the same donor as used. It is possible that theagarose culture wells limited the diffusion of ECM molecules produced bycells into the bulk medium, thereby increasing their effectiveconcentration in the constructs and the probability of macromoleculeassembly and matrix maturation. For example, aggrecan, acartilage-specific proteoglycan which plays an important role inresisting cartilage compression during loading, is noncovalently boundto hyaluronic acid and stabilized by link protein to form large aggrecanaggregates outside the cell. Collagen fiber bundles are also assembledextracellularly. Additionally, proteoglycane collagen interaction isessential for cartilaginous ECM function. A potentially similarbiophysical approach called macromolecular crowding, which incorporateslarge molecules as a means of increasing medium density and limitingdiffusion, has been shown to drastically increase deposition of type Icollagen by fibroblasts in tissue culture. Another possibility forimproved chondrogenesis is the increased surface area to volume ratio ofthe toroid compared to sheet culture for the same number of cells, whichcould result in greater availability of oxygen and nutrients and betterremoval of waste via diffusion.

The custom well and assembly system in this Example was used to engineera tracheal replacement which can be initially tested in a small animalmodel for tracheal defects in a rat. With regard to tissue dimensions,rat tracheas have a similar lumen diameter but the walls of engineeredrings were significantly thicker (FIG. 11A) and tubes had significantlygreater outer diameters (FIG. 11B) compared to rat tracheas. Mechanicalevaluation by uniaxial UTS on the rings and luminal collapse and recoilon the tubes showed that scaffold-free cartilaginous rings and tubesperform at least as well as native rat trachea, suggesting that theseengineered tissues may be able to provide the mechanical rigiditynecessary to maintain airway patency in the rat. It is promising thatthe tissue engineered microsphere-containing neocartilaginous tubesrequired significantly greater loads to collapse the lumen compared tothe similarly-sized rat tracheal segments because a trachea for clinicaluse in humans will likely need to be stiffer than a rat trachea. Whiletissue engineered cartilage rings and tubes appeared thicker than rattracheal cartilage, they are very similar to the thickness of humantracheal cartilage rings. Unlike the tissue engineered torus rings witha circular cross-section presented here, human tracheal cartilage ringsare toroid-like with a more rectangular-shaped cross-sectional areawhich is typically about 1 mm radially and about 4 mm vertically.Control over the vertical dimension of engineered tubes can be achievedby fusing multiple rings together, as shown in this Example. A longerengineered trachea would simply require more cells, microspheres, growthfactor and media, but tissue generation should not be inhibited by thelength of the construct. Adult human trachea also has a much largerlumen, measuring at least 12 mm in diameter, but using this approach itwill be possible to engineer larger diameter rings and tubes bymodifying the size of the cell culture annular wells for ringself-assembly and the support strut for tube culture. Additionally, thegeometry of the culture-wells and the tissue assembly approach can beeasily altered to produce self-assembled tissues of specific shapes(e.g., oval tissues with defined wall thickness, cone like structures,or even figure-eight, honeycomb and dog bone shaped constructs) forapplications necessitating geometrical control over anatomical featuresand/or tissue-level morphology.

A functional tracheal replacement may require much more complexity intissue organization and function. While the cartilaginous portion of thenative trachea provides support to the airway, intervening vascularizedfibrous tissue is necessary to supply the cartilaginous rings andmucosal and submucosal layers lining the tracheal lumen with nutrientsand oxygen. Our customizable tissue assembly system may permit theintegration of these vital tissue components to replicate actualtracheal architecture and ultimately function. Firstly, donor-specificneeds with regard to tissue anatomy may be addressed by employingannular wells and support struts with custom geometry to engineer theorgan. Next, different cell sources and/or differentiation conditionsfor the tissue units can be used to engineer tissues with requisiteproperties, such as rings with neovasculogenic capabilities or tubes oftracheal epithelium. Thirdly, incorporation of bioactive microsphereswith different compositions into each type of tissue ring may allow forspatial as well as temporal control of cell differentiation andneotissue formation even after multi-tissue fusion. It is also possiblethat incorporation of growth factor-loaded microspheres can decrease invitro culture time by releasing bioactive factors after implantation,and in doing so stimulate in vivo tissue maturation and physiologicalhealing. The use of bioactive microspheres in the modular custom culturesystem described here is a promising approach for tracheal tissueregeneration.

Example 2

This Example shows the generation of osteognic rings and tubes from hMSCthat include TGF-β1 and BMP-2 loaded nanoparticles.

hMSC Isolation and Expansion

Human mesenchymal stem cells (hMSCs) were isolated from the posterioriliac crest of 3 healthy male donors (43 ± 5 years) using a protocolapproved by the University Hospitals of Cleveland Institutional ReviewBoard and cultured as previously described. Briefly, the aspirates wererinsed with low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG;Sigma-Aldrich, St. Louis, MO) with 10 % prescreened fetal bovine serum(Sigma-Aldrich). Mononucleated cells were isolated using a Percoll(Sigma-Aldrich) density gradient then plated on tissue culture plasticat a density of 1.8 × 10⁵ cells per cm² in medium containing 10 ng/mlfibroblast growth factor-2 (FGF-2; R&D Systems, Minneapolis, MN) andcultured at 37° C. with 5% CO₂. Nonadherent cells were removed after 4days. The adherent cells, primary hMSCs, were cultured for another 10-14days with media changes every 3 days. They were then reseeded at 4 × 10³cells/cm² and expanded until passage 2, when they were stored in liquidnitrogen in DMEM-LG with 10 % dimethyl sulfoxide (DMSO) until use.

Gelatin Microsphere (GM) Synthesis and TGF-β1 Loading

Gelatin microspheres (GM) were synthesized using a water-in-oil singleemulsion technique and crosslinked with genipin for 2 hours aspreviously described. Briefly, 11.1 % w/v acidic gelatin (Sigma-Aldrich)was dissolved in deionized water (diH₂O), added drop-wise into 250 mlpreheated (45° C.) olive oil (GiaRussa, Coitsville, OH) and magneticallystirred at 500 RPM. After 10 minutes, stirring ensued at 4° C. for 30minutes. 100 ml acetone chilled at 4° C. was added to the emulsion andagain an hour later. Stirring rate increased to 1000 RPM for 5 minutesafter which the solution was filtered and the resulting microsphereswere washed with acetone and dried overnight. Microspheres were thencrosslinked with 1% w/v genipin (Wako USA, Richmond, VA) on a magneticstir plate at RT. After 2 hours, the crosslinked microspheres wererinsed 3 times with diH₂O and lyophilized. Characterization of GM can befound in Solorio et al. 2012. Prior to adding the microparticles to thehMSC suspension, empty GM were incubated with PBS for 2 hours at 37° C.For exogenous growth factor supplementation, TGF-β1 (Peprotech, RockyHill, NJ) was added to the medium at 10 ng/ml.

Mineral-Coated Hydroxyapatite Microparticle (MCM) Synthesis and BMP-2Loading

Hydroxyapatite (HA) microparticles ranging from 3-5 µm in diameter fromPlasma Biotal LTD (Derbyshire, UK) were mineral-coated in modifiedsimulated body fluid (mSBF) and loaded with BMP-2 as previouslydescribed. Briefly, HA microparticles were added at 2 mg/ml to mSBF (pH6.8) containing 141 mM NaCl, 4.0 mM KCl, 0.5 mM MgSO₄, 1.0 mM MgCl₂,20.0 mM HEPES, 5.0 mM CaCl₂, 2.0 mM KH₂PO₄ and 4.2 mM NaHCO₃ (all fromFisher Scientific) The solution was stirred continuously at 37° C. for 7days with the mSBF refreshed daily. At the end of the coating process,the MCMs were rinsed with diH₂O and lyophilized. Prior to adding themicroparticles to the hMSC suspension, empty MCM were incubated with PBSfor 4 hours at 37° C. For exogenous growth factor supplementation, BMP-2(Dr. Walter Sebald, Department of Developmental Biology, University ofWürzburg, Germany) was added to the medium at 100 ng/ml.

Cell Culture Well Preparation

Agarose molds for cell culture were prepared as previously described.Briefly, a polycarbonate sheet (Small Parts Inc., Miramar, FL) wasmachined to contain annular wells with concentric 2 mm diameter postssurrounded by a 3.75 mm wide trough. A polydimethylsiloxane (PDMS;Sylgard 184, Dow Corning) negative mold of the polycarbonate templatewas cured and sterilized. Two percent w/v agarose (Denville ScientificInc., Metuchen, NJ) in DMEM-LG (Sigma-Aldrich) was autoclaved and usedto fill the PDMS mold. After cooling, the ring-shaped culture wells wereremoved from the PDMS mold, moved into 6-well plates (BD Falcon), andincubated overnight in a serum-free, chemically-defined basal pelletmedium (BPM) containing DMEM-HG (Sigma-Aldrich) with 10% ITS+ Premix(Corning), 1 mM sodium pyruvate (HyClone), 100 µM non-essential aminoacids (Lonza), 100 nM dexamethasone (MP Biomedicals, Solon, OH), and0.05 mM L-ascorbic acid-2-phosphate (Wako).

Assembly of Microsphere-containing Tissue Rings and Tubes

Trypsinized hMSCs (400,000 cells) with or without 0.3 mg GM and 0.08 mgMCM in 50 µL media were seeded in a circular fashion in each customdesigned annular well and cultured in BPM + 10 ng/ml TGF-β1. After 24hours, agarose wells were flooded with media. On day 2, theself-assembled rings were transferred from the annular wells onto 2-mmglass tubes (Adams & Chittenden Scientific Glass, Berkeley, CA) asindividual 2-mm rings, or to form 3×2-mm and 8×2-mm tubes. Glass tubeswere placed on top of custom engineered polycarbonate holders and tissuerings/tubes were cultured horizontally in 60 mm petri dishes (BD Falcon)in a humidified cell culture incubator at 37° C. and 5% CO₂ for 2 weeksin chondrogenic induction medium (BPM + 10 ng/ml TGF-β1) followed by 3weeks in osteogenic induction medium comprised of DMEM-HG(Sigma-Aldrich) with 10% ITS+ Premix (Corning), 1 mM sodium pyruvate(HyClone), 100 µM non-essential amino acids (Lonza), 100 nMdexamethasone (MP Biomedicals, Solon, OH), 0.173 mM L-ascorbicacid-2-phosphate (Wako), and 5 mM β-glycerophosphate (Sigma-Aldrich) +100 ng/ml BMP-2. The induction media were changed every 3 days.

Gross Morphological Assessment

After 5 weeks, rings and tubes were harvested and gross images of alltissues were taken. Thickness and length measurements were performedusing micro calipers (Fowler). Four measurements were obtained perspecimen at the 12, 3, 6, and 9 o′clock positions.

Statistical Analysis

All data are expressed as mean ± SD. The unpaired Student’s t test wasused to test for significant effects with p<0.05 considered significant.Data were analyzed using GraphPad Prism 6.0 software (GraphPad SoftwareInc., La Jolla, CA).

Results Morphological Assessment

The gross morphology of the rings and tubes was visually assessed after5 weeks of chondrogenic and osteogenic induction in presence of TGF-β1(2 weeks) and BMP-2 (3 weeks). Tissue rings and tubes comprised of hMSCsonly were noticeably thinner compared to hMSC + GM + MCM specimens. Incontrast, the overall length of the hMSC + GM + MCM rings and 3×2-mmtubes appeared reduced, while the 8×2-mm tubes had comparable lengthsacross groups (FIGS. 14 ).

Quantitative thickness and length analyses of the rings and tubes werein agreement with the qualitative assessment. Rings containing GM + MCMwere significantly thicker than hMSC rings alone (FIG. 15A; p<0.05).Both tissue tubes (3×2-mm and 8×2-mm) revealed similar trends (FIGS.15C, E; p<00.001). In contrast, measurements of the hMSC + GM + MCMrings showed a significant length reduction (FIG. 15B; p<0.05) comparedto the hMSC rings alone, which was confirmed by the 3×2-mm tubes (FIG.15D; p<0.001). No differences were observed in the length of 8×2-mmtubes across groups (FIG. 15F).

Example 3

This Example shows the generation of vascular rings and tubes fromsmooth muscle cells that include TGF-β1 loaded microparticles.

FIGS. 16 illustrates a schematic view of microsphere incorporationwithin self-assembled cell rings. A, Cross-linked gelatin microsphereswere mixed in suspension with smooth muscle cells at 0, 0.2 or 0.6 mgmicrospheres per million cells. B, cells and microspheres were seededinto agarose molds. Cells aggregate to form self-assembled tissue ringswith incorporated microspheres (C). D, Photograph of an agarose moldwith three aggregated cell-microsphere rings cultured for 14 days.Arrowheads point to self-assembled tissue rings on agarose posts (2 mmpost diameter).

FIGS. 17 illustrates images and a graph showing microspheres increasedtissue ring thickness. Images of self-assembled cell rings seeded with 0(A), 0.2 (B), or 0.6 mg (C) of microspheres per million cells andcultured in smooth muscle growth medium for 14 days. (D) Average wallthicknesses of 14-day-old tissue rings with 0, 0.2, or 0.6 mgmicrospheres per million cells. Scale = 1 mm, n = 6, *p<0.05.

FIGS. 18 illustrates images showing gelatin microsphere incorporationand degradation within tissue rings. Tissue rings were seeded with 0,0.2, or 0.6 mg microspheres per million cells, collected at 7 or 14days, and stained with Hematoxylin and Eosin (A-F) and PicrosiriusRed/Fast Green stain. Scale = 100 µm.

FIGS. 19 illustrates graphs showing Mechanical properties of tissuerings loaded with gelatin microspheres. Self-assembled cell rings werecultured for 14 days in growth medium and pulled to failure. Mean valuesfor ultimate tensile strength (UTS; A), maximum tangent modulus (MTM;B), failure load (C) and failure strain (D) were calculated fromstress-strain curves for each ring sample group. n = 6, *p<0.05.

FIGS. 20 illustrates an image and graph showing microspheres increasedring thickness in tissues cultured in smooth muscle differentiationmedium. Rings were seeded with 0 (A), 0.2 (B), or 0.6 mg (C) ofmicrospheres per million cells and cultured to 14 days. Rings wereseeded in growth medium and switched to differentiation medium on day 1.(D) Average wall thicknesses of 14-day-old tissue rings with 0, 0.2, or0.6 mg microspheres per million cells. Scale = 1 mm; n = 8 for 0 mg; n =9 for 0.2 and 0.6 mg/million cells; *p<0.05.

FIGS. 21 illustrates images showing microsphere incorporation withintissue rings cultured in smooth muscle differentiation medium. Tissuerings were seeded in growth medium with 0, 0.2, or 0.6 mg microspheresper million cells, and switched to differentiation medium at day 1.Tissue rings were collected at 7 or 14 days, and stained withHematoxylin and Eosin (A-F) and Picrosirius Red/Fast Green stain. Scale= 100 µm.

FIGS. 22 illustrates graphs showing the mechanical properties of tissuerings cultured in differentiation medium with microsphere incorporation.Self-assembled cell rings were seeded in growth medium, switched todifferentiation medium on day 1, and cultured for 13 days indifferentiation medium and harvested for mechanical tests (14 days totalculture). Mean values for ultimate tensile strength (UTS; A), maximumtangent modulus (MTM; B), failure load (C) and failure strain (D) werecalculated from stress-strain curves for each ring sample group. n = 6,*p<0.05.

FIGS. 23 illustrates images and a graph showing exogenous ormicrosphere-mediated TGF-β1 delivery to self-assembled tissue rings.Rings were seeded in growth medium, and switched to differentiationmedium at day 1. (A) Untreated control rings with no microspheres (n=6).(B) Tissue rings treated with 10 ng/ml soluble TGF-β1 (n=8). (C,D)Tissue rings with unloaded gelatin microspheres (0.6 mg/million cells;n=6) untreated (C) or treated (D) with 10 ng/ml exogenous TGF-β1 (n=8).(E) Tissue rings with microspheres pre-loaded with TGF-β1 (400 ngTGF-β1/mg microspheres), but no exogenous TGF-β1 in the medium (n=7).Tissue rings contracted after they were removed from agarose posts,resulting in a greater decrease in diameter (F) and greater thickness(G) in rings exposed to TGF-β1. Scale = 1 mm, *p<0.05.

FIGS. 24 illustrates images showing hematoxylin and eosin stain (A-E) at14 days shows microsphere degradation primarily in the groups with addedTGF-β1. Collagen deposition (F-J, Picrosirius Red/Fast Green stain,red=collagen green=counterstain) in TGF-β1 groups is primarily seenaround ring edges. (A,F) Control (untreated) rings. (B,G) Rings culturedwith exogenous 10 ng/ml TGF-β1 added to the medium. Rings with unloadedmicrospheres (0.6 mg per million cells) untreated (C,H) or treated with10 ng/ml exogenous TGF-β1 (D,I). Rings with TGF-β1 loaded microspheres(0.6 mg microspheres per million cells) and no exogenous TGF-β1treatment (E, J). Scale = 100 µm.

FIGS. 25 illustrates images showing Contractile protein expression intissue rings treated with TGF-β1. Rings were grown with either nomicrospheres or exogenous TGF-β1 (A,F), no microspheres but treated with10 ng/ml exogenous TGF-β1 (B,G), with microspheres and no exogenousTGF-β1 (C,H), with unloaded microspheres and exogenous TGF-β1 (D,I) orwith TGF-β1 loaded microspheres and no exogenous TGF-β1 in the medium(E,J). Rings were stained for either smooth muscle alpha actin (A-E) orcalponin (F-J). Nuclei are shown in blue (Hoechst). Scale = 100 µm.

FIGS. 26 illustrates graphs showing the mechanical properties of tissuerings treated with TGF-β1 after 14 days in culture. Rings were culturedin differentiation medium with no microspheres or exogenous TGF-β1, nomicrospheres with 10 ng/ml exogenous TGF-β1, unloaded microspheres withno exogenous TGF-β1, unloaded microspheres with 10 ng/ml exogenousTGF-β1, or loaded microspheres (400 ng TGF-β1/mg microspheres) and noexogenous TGF-β1. Rings in the group with no microspheres and exogenousTGF-β1 had significantly higher ultimate tensile stresses than theloaded microsphere group (A), and the unloaded microspheres withoutTGF-β1 group had significantly higher failure loads than rings withoutmicrospheres or TGF-β1 (B). There were no significant differences in MTM(C) or failure strain (D). *p<0.05.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes, and modifications are within the skill of the artand are intended to be covered by the appended claims. All patents andpublications identified herein are incorporated by reference in theirentirety.

Having described the invention we claim: 1-10. (canceled)
 11. A modularengineered tissue construct, comprising; a plurality of fusedself-assembled, scaffold-free, high-density cell aggregates, wherein atleast one cell aggregate includes a plurality of cells and a pluralityof biocompatible and biodegradable nanoparticles and/or microparticlesthat are incorporated within the cell aggregates, the nanoparticlesand/or microparticles acting as a bulking agent within the cellaggregate to increase the cell aggregate size and/or thickness andimprove the mechanical properties of the cell aggregate.
 12. The modularengineered tissue construct of claim 11, wherein the self-assembled,scaffold-free, high-density cell aggregates comprise differing aggregatematerials, at least one of the ring-shaped self-assembled,scaffold-free, high-density cell aggregates being provided or formedwith or without nanoparticles and/or microparticles and having differentproperties than the other aggregates to vary the properties of theconstruct for particular tissue engineering applications.
 13. Themodular engineered tissue construct of claim 11, wherein the at leastone of the fused ring-shaped self-assembled, scaffold-free, high-densitycell aggregates comprises a plurality of chondrogenic cells that havebeen differentiated to form engineered cartilage.
 14. The modularengineered tissue construct of claim 11, comprising alternating firstengineered ring-shaped self-assembled, scaffold-free, high-density cellaggregates and second engineered ring-shaped self-assembled,scaffold-free, high-density cell aggregates fused to form a heterogenousmodular tissue tube.
 15. The engineered modular tissue construct ofclaim 14, wherein the first engineered ring-shaped self-assembled,scaffold-free, high-density cell aggregates define cartilaginousportions within the tube and the second engineered ring-shapedself-assembled, scaffold-free, high-density cell aggregates definenoncartilaginous portions within the tube.
 16. The engineered modulartissue construct of claim 11, the nanoparticles and/or microparticlescomprising a biocompatible and biodegradable polymer.
 17. The engineeredmodular tissue construct of claim 11, the nanoparticles and/ormicroparticles including at least one bioactive agent that isdifferentially and/or controllably released by the nanoparticles and/ormicroparticles.
 18. The engineered modular tissue construct 17,bioactive agent including at least one of TGF-β1 and/or BMP-2. 19-28.(canceled)
 29. An engineered trachea implant comprising: a plurality offused ring-shaped self-assembled, scaffold-free, high-density cellaggregates, at least one cell aggregate including a plurality of cellsand a plurality of biocompatible and biodegradable nanoparticles and/ormicroparticles that are incorporated within the cell aggregates, thenanoparticles and/or microparticles acting as a bulking agent within thecell aggregate to increase the cell aggregate size and/or thickness andimprove the mechanical properties of the cell aggregate.
 30. Theengineered trachea implant of claim 29, wherein the ring-shapedself-assembled, scaffold-free, high-density cell aggregates comprisediffering aggregate materials, at least one of the ring-shapedself-assembled, scaffold-free, high-density cell aggregates beingprovided or formed with or without nanoparticles and/or microparticlesand having different properties than the other aggregates to vary theproperties of the tube for particular tissue engineering application.31. The engineered trachea implant of claim 29, wherein the at least oneof the fused ring-shaped self-assembled, scaffold-free, high-densitycell aggregates comprises a plurality of chondrogenic cells that havebeen differentiated to form engineered cartilage.
 32. The engineeredtrachea implant of claim 29, comprising alternating first engineeredring-shaped self-assembled, scaffold-free, high-density cell aggregatesand second engineered ring-shaped self-assembled, scaffold-free,high-density cell aggregates fused to form a heterogenous modular tissuetube.
 33. The engineered trachea implant of claim 29, wherein the firstengineered ring-shaped self-assembled, scaffold-free, high-density cellaggregates define cartilaginous portions within the tube and the secondengineered ring-shaped self-assembled, scaffold-free, high-density cellaggregates define noncartilaginous portions within the tube.
 34. Theengineered trachea implant of claim 29, the nanoparticles and/ormicroparticles comprising a biocompatible and biodegradable polymer. 35.The engineered trachea implant of claim 29, the nanoparticles and/ormicroparticles including at least one bioactive agent that isdifferentially and/or controllably released by the nanoparticles and/ormicroparticles.
 36. The engineered trachea implant of claim 29,bioactive agent including at least one of TGF-β1 and/or BMP-2.
 37. Theengineered trachea implant of claim 29, wherein an inner lumen of theimplant includes a mucosal epithelial lining.